Use of cell lines to produce active therapeutic proteins

ABSTRACT

This invention relates to the production of proteins, including therapeutic plasma proteins, by virally-immortalized hepatocyte cell lines and by other eukaryotic cells, as well as to the use of such proteins for screening and therapy, as well as to nucleic acids, vectors, and transformed or transfected cells that carry the genetic information for proteins.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/510,509, filed on Oct. 10, 2003, which is hereby incorporated inits entirety by reference.

GOVERNMENT GRANTS

This invention was made in part with United States government supportunder grant number 70-NANB7H3070 awarded by Advanced Technology Programof the United States Department of Commerce and with support from a NIHSmall Business Innovative Research grant (Grant number: R 1 43GM66480-01). The U.S. government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the use of cell lines, particularlyvirally-immortalized normal human cell lines, to produce proteins,especially therapeutic proteins, including therapeutic plasma proteins(TPP), that are capable of being expressed in active form by hepatocytesand to the use of proteins, therapeutic proteins, and especially plasmaproteins, produced by hepatocytes for the treatment of diseases andconditions affecting the liver and other organs.

BACKGROUND OF THE INVENTION

The safe and efficient production of novel therapeutic proteinsrepresents an expanding market of the biopharmaceutical industry that isfueled by the recent completion of the Human Genome Project and by rapidtechnological advances in the field of proteomics. Paulaus, A., Thereengineering of drug development in the genomics and proteomics era. AmClinical lab, 2001. 5: p. 55-57.

Although many of these therapeutic proteins are mass-produced byrecombinant technology in Chinese Hamster Ovary (CHO) cells and othernon human cell types, there are occasions where the commercialization ofcomplex heterologous proteins is better accomplished by using the nativeform of the therapeutically effective protein. This is particularly truewhen such proteins are the products of multiple genes and the resultingproteins are highly processed post-translationally. It follows that thismay be accomplished by isolating the native form of the protein or arecombinant form of the protein that is expressed and processed in thehuman producer cell.

In addition, the production of therapeutic plasma proteins (TPP) bycell-based systems would avoid the hazards of blood-derived products,the most notable of which is viral contamination. Although, whenprocessed correctly, blood-derived products are virtually free oftransmitting viral infections, a perceived risk exists for themanufacturer, user, and patient. Indeed, the recent discovery of newstrains of human immunodeficiency virus and the agents responsible forthe transmissible spongiform encephalopathies, such as mad cow disease,exemplify the everlasting concern for blood-derived products. Collinge,J., et al., Molecular analysis of prion strain variation and theaetiology of ‘new variant’ CJD. Nature, 1996. 383(6602): p. 685-90.

Limitations of Recombinant Proteins as Therapeutic Drugs.

Currently, many proteins that have been approved for clinical andtherapeutic use, with the exception of monoclonal antibodies, aremass-produced by recombinant protein technology. Although these productshave been proven safe and effective, not all behave identically to theirnative counterparts. For example, recombinant factors blood clottingfactors (rF) VIII and IX are more rapidly cleared following infusionthan their plasma derived counterparts. Shapiro, A., E. Berntorp, and M.Morfini, Incremental recovery assessment and effects of weight and agein previously untreated patients treated with recombinant factor IX.Blood, 2000. 96 (suppl 1): p. 265a

Recent findings suggest that this is the result of incomplete orinappropriate post-translational modification. The rapid clearance ofthe β-domain deleted form of Factor VIII, which is used in the UnitedStates, is due to differences in phospholipid binding. In contrast,differences in sulfation at tyrosine 155 and phosphorylation of serine158 of Factor IX result in more rapid clearance of the clotting factor.White, C. G. I., A. Beebe, and B. Nielsen, Recombinant factor IX. ThrombHaemost, 1997. 78: p. 261-265.

Clinically, more rapid clearance of these clotting factors meanspotentially more frequent and higher dosages depending upon the patientpopulation. Although one strategy to circumvent these shortcomings is touse plasma-derived proteins, there are also perceived risks, asmentioned above, associated with this approach.

Significant Unmet Need for Therapeutic Proteins

Hemophilia A (Factor VIII deficiency) and hemophilia B (Factor IXdeficiency) are bleeding disorders that are inherited as X-linkedrecessive traits. Thus, both affect males almost exclusively. Bothhemophilia A and hemophilia B are heterogeneous conditions with variabledegrees of clinical expression. Hemophilia A is far more common,occurring in 1 in 5000 to 10,000 males in the United States. Soucie, J.M., B. Evatt, and D. Jackson, Hemophilia Occurrence in the UnitedStates. American Journal of Hematology, 1998. 59: p. 288-294.

In contrast, the incidence of hemophilia B is 0.25 in 10,000 males.Currently, plasma-derived and recombinant Factor VIII and IXconcentrates are used for the lifetime treatment of hemophilia. It isestimated that three-quarters of the worldwide hemophilia populationreceive little or no treatment due to a shortage of this TPP. Thus,there is a clear need for fully functional, naturally-processedblood-clotting factors to overcome the shortcomings of traditionalrecombinant methodologies and/or the limited availability ofblood-derived TPPs.

α-1-antitrypsin (AAT) is a human blood protein whose prime physiologicaltarget is neutrophil elastase. Severe AAT deficiency (hereditaryemphysema) is thought to affect around 150,000-200,000 individuals inEurope and US. Donohue, T. M., et al., Synthesis and secretion of plasmaproteins by the liver, in Hepatology: A Textbook of Liver Disease, D.Zakim and T. D. Boyer, Editors. 1990, W.B. Sounders Company:Philadelphia p. 124-137. Many respiratory diseases including AATcongenital deficiency, cystic fibrosis, and chronic obstructivepulmonary disease are characterized by an imbalance of AAT and elastasein the lung. Elastase is a serine protease that hydrolyzes theextracellular matrix protein molecule elastin, among other proteins. Anabundance of elastase is thought to contribute to damage of thepulmonary epithelium.

Administration of supplemental AAT is therefore expected to alleviatethe deleterious effects of elastase in the lung in these diseases.

Approximately one in 2000 children is born with the CF genetic defect inthe Western Hemisphere. Currently, there is only one plasma-derived AATlicensed in the United States, which has been in very limited supply.Many of the diagnosed patients have therefore not had access to AATtreatment. Despite the large body of evidence of the clinical efficacyof AAT to treat general inflammatory conditions, its use has beenrestricted due to the limited availability of the product. Thus, thereis a clear need for fully functional, naturally-processed AAT toovercome the shortcomings of recombinant or blood-derived TPPs.

Sepsis is a disease characterized by an overwhelming systemic responseto infection, which can rapidly lead to organ dysfunction and ultimatelydeath. Sepsis can strike anyone and can be triggered by events such aspneumonia, trauma, surgery and burns, or by conditions such as cancer orAIDS. Once triggered, an uncontrolled cascade of coagulation, impairedfibrinolysis (clot breakdown), and inflammation fuels the progression ofsepsis. In the United States, sepsis is the leading cause of death inthe noncardiac intensive care unit and the 11^(th) leading cause ofdeath overall.

Each year, over 700,000 new cases of sepsis are diagnosed and every day1400 people worldwide die from severe sepsis. Currently, treatment forsepsis is limited to attempts to manage the underlying infection andsupportive therapy if the infection leads to organ dysfunction. Despiteintensive medical care, up to 50% of patients still die from thisillness. Rangel-Frausto, M. S., et al., The natural history of thesystemic inflammatory response syndrome (SIRS): a prospective study.JAMA, 1995. 273: p. 117-123.

Given the intensive and prolonged care necessary to treat patients withsepsis, the economic burden is profound. For decades, physicianstreating patients with severe septic illness have searched for aneffective addition to their available therapeutic arsenal (mainlyantibiotics) that could reduce the high mortality rate associated withthis disease. Many of the attempted therapeutic interventions in humansepsis have been based upon the premise that circulating endotoxin isresponsible for the critical clinical manifestations and morbidity ofsepsis. Indeed, some investigators have concluded that any adjunctivetherapy is destined to fail because once the clinical signs of severesepsis are present, irreversible organ injury has already occurred.Recently, a promising new class of therapeutic agents based on naturalplasma proteins with anti-coagulative activities has appeared on theclinical horizon. In severe sepsis, the coagulation system is activated;an event evidenced by the presence of intravascular thrombi in vesselsand tissue and the occurrence of disseminated intravascular coagulation.Large multicenter phase III studies of activated protein C (APC) andantithrombin III (AT-III) in sepsis were completed in early 2001. Inlate 2001, Eli Lilly began marketing, Xigris, a genetically engineeredversion of the human activated protein C molecule; however, this drugonly reduces the absolute risk of death by six percent. There is a clearneed for more effective treatments of this severe sepsis.

Inter-α-inhibitor proteins (IαIp), natural serine protease inhibitorsfound in relatively high concentration in plasma have been shown to playroles in inflammation, wound healing and cancer metastasis reviewed byBost et al. Bost, F., M. Diarra-Mehrpour, and J. P. Martin,Inter-alpha-trypsin inhibitor proteoglycan family—a group of proteinsbinding and stabilizing the extracellular matrix. Eur J Biochem, 1998.252: p. 339-346. The major forms of IαIp are inter-α-inhibitor (IαI,containing one light chain peptide called bikunin and two heavy chains)and pre-α-inhibitor PαI, containing one light chain and one heavychain). In IαI, the two heavy chains are designated H1 and H2.

These are cleaved from precursors designated H1P and H2P. Theseprecursors are encoded by genes designated ITH1 and ITH2. The bikuninsubunit is a double-headed, Kunitz-type, protease inhibitor. It isproduced by cleavage from a α1m/bikunin precursor known as AMBP andencoded by a gene designated AMBP. The properties of the AMBP fusionprotein precursor and the cleavage process are discussed further below.In PαI, the light chain is bikunin, and the heavy chain is H3, cleavedfrom a precursor designated H3P, encoded by a gene designated ITIH3.Both IαI and PαI are designated IαIp protein complexes, and that term isused herein to refer generally to either of IαI and PαI, or to both IαIand PαI.

Recently, a monoclonal antibody that recognizes the light chain of humanIαIp (MAb 69.31) was developed by scientists at Prothera Biologics(Providence, R.I.). Using MAb 69.31 in a competitive ELISA, theseinvestigators demonstrated that plasma IαIp levels were significantlydecreased in severe septic patients compared to healthy controls. Thisdecrease correlated with mortality suggesting that IαIp might havepredictive value in septic patients. Lim, Y. P., et al., Inter-trypsininhibitor: decreased plasma levels in septic patients and its beneficialeffects in an experimental sepsis model. Shock, 2000. 13 (Suppl.): p.161. In-vivo animal studies using a polymicrobial sepsis rat model ofcecal ligation and puncture showed that administration of IαIp produceddramatic improvements in survival rates compared to saline controls.Yang S, et al., Administration of human inter-alpha-inhibitors maintainshemodynamic stability and improves survival during sepsis. Crit CareMed. March 2002; 30(3):617-22. Taken together, the results stronglysupport the therapeutic potential of IαIp in the management of severesepsis. Although IαIp can be purified from human serum or plasma, ifproven effective there will remain a worldwide shortage of this proteinto treat sepsis. There is presently no means to produce or expresshighly functional, naturally-processed forms of this IαIp (e.g.naturally occurring or recombinant produced). Further the complexity ofthis protein increases the difficulty of both expressing it in anactive, processed form and isolating it in an active state.

There are a number of patents and publications that describeimmortalized cell lines: U.S. Pat. No. 6,107,043 (Jauregui); U.S. Pat.No. 5,665,589 (Harris); U.S. Patent App. No. 2002/0045262 A1(Prachumsri); and International publication No. WO 99/55853 (Namba).However, to date, among other things, the prior art cell lines do notprovide a means to safely, effectively, and cost efficiently perform theprotein post-translational modifications, such as glycosylation, thatare critical in the production of functional therapeutic plasma protein;produce simultaneously multiple therapeutic plasma proteins, especiallyFactor VIII protein or Factor IX, as well as IαIp; and maintain thecontinuous expression of active levels of cytochrome P450 enzyme in aserum-free media.

SUMMARY OF THE INVENTION

One aspect of the present invention is a method of using immortalizedhuman hepatocyte cells to produce a protein comprising the steps of:

(1) providing an immortalized human hepatocyte cell that includes DNAthat encodes and can express a protein;

(2) culturing the immortalized hepatocyte cell under conditions in whicha gene or genes encoding the protein are expressed so that the proteinis produced and processed in the immortalized hepatocyte cell; and

(3) isolating the processed protein from the immortalized hepatocytecell; wherein the protein is expressed such that the protein isprocessed and glycosylated, if necessary, so that its in vivo functionis substantially preserved after its isolation.

The protein can be a protein that is naturally produced by humanhepatocytes, or can be a protein that is not naturally produced by humanhepatocytes. Preferably the protein is a therapeutic protein. Morepreferably, the therapeutic protein is a plasma-derived therapeuticprotein.

The protein can also be a mutein of a protein that is normally producedby human hepatocytes.

The protein can be a therapeutic protein, such as a plasma proteinselected from the group consisting of Factor VIII, Factor IX, humangrowth hormone (hGH), α-1-antitrypsin, transferrin, and a growth factor,or a mutein of one of these proteins.

Alternatively, the protein can be a protein selected from the groupconsisting of albumin, transcobalamin II, C-reactive protein,fibronectin, ceruloplasmin, and other proteins having structural,enzymatic, or transport activities, or a mutein of one of theseproteins.

The protein can be one that is expressed by a gene that occurs naturallyin the hepatocytes, in which case expression of the naturally-occurringgene encoding the protein is enhanced by introduction of a high-levelpromoter into the hepatocytes.

Alternatively, expression is enhanced by introducing multiple copies ofthe gene encoding the protein to be expressed, a subunit of the proteinto be expressed, or a precursor of the protein to be expressed via theuse of one or more recombinant vectors that include: (1) the geneencoding the protein to be expressed, a subunit of the protein to beexpressed, or a precursor of the protein to be expressed; and (2) atleast one control element affecting the transcription of the gene, thecontrol element being operably linked to the gene.

In one alternative, the expressed protein is secreted from the cell intothe surrounding culture medium.

The protein can be glycosylated or post-translationally processed.

The protein can be expressed in a form wherein it is fused to acleavable tag.

The protein comprises at least two different subunits. In this case, theimmortalized hepatocyte cell can be transformed or transfected with atleast two vectors, each vector including: (1) DNA including at least onegene that encodes at least one subunit of the protein; and at least onecontrol element operably linked to the DNA encoding at least one genethat encodes the subunit of the protein.

Another aspect of the invention is a method of treating a disease orcondition comprising the steps of:

(a) providing an active protein produced by the method described above;and

(b) administering the active protein to a patient suffering from thedisease or condition in a therapeutically effective quantity to treatthe disease or condition.

Preferrably, the protein produced by the methods of the presentinvention are formulated in a pharmaceutical composition for delivery tothe patient suffering from the disease or condition.

Another aspect of the invention is a method of using eukaryotic cells,other than human hepatocytes, to produce an IαIp protein complexcomprising the steps of:

(1) providing a eukaryotic cell, other than a human hepatocyte, thatincludes DNA that encodes and can express proteins forming an IαIpprotein complex, the eukaryotic cell having been transformed ortransfected with at least one vector that includes: (a) DNA including atleast one gene for a precursor of a protein that is part of an IαIpprotein complex; and (b) at least one control element operably linked tothe DNA encoding at least one precursor gene in order to enhanceexpression of the precursor gene;

(2) culturing the transformed or transfected eukaryotic cell underconditions in which genes encoding proteins forming an IαIp complex areexpressed so that an IαIp complex is produced; and.

(3) isolating the expressed IαIp protein complex from the transformed ortransfected eukaryotic cell.

Another aspect of the invention is an immortalized human hepatocyte cellthat includes DNA that encodes and can express a protein, theimmortalized human hepatocyte cell having been transformed ortransfected with at least one vector that includes: (1) DNA including atleast one gene encoding a protein; and (2) at least one control elementoperably linked to the DNA encoding the protein in order to enhanceexpression of the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows immunostaining of the Ea1C-35 immortalized hepatocytecell line for large T antigen that confirms the integration of SV40DNAinto genomic DNA of the immortalized cell.

FIG. 1 b shows immunostaining of cultured Fa2N-4 cells that demonstratesthat the proliferating cells continue to express albumin.

FIG. 1 c shows the morphology of the immortalized cells showingwell-defined nucleoli and granulated cytoplasm, which are characteristicfeatures of normal primary hepatocytes.

FIG. 2 shows an immunoblot showing induction of CYP3A4 consequent oftreatment of Fa2N-4 and EA1C-35 with Rifampin (RIF), beta-naphthoflavone(BNF) and phenobarbital (PB). C is the untreated control. It should benoted that the upper band is nonspecific and that BNF, a CYP1A inducerdoes not induce CYP3A4 protein expression.

FIG. 3 shows the following lanes: 1) Human Plasma; 2) Empty, 3) CultureMedium (Control); 4) Primary human hepatocytes (72 hr culture); 5)Ea1C-35 monolayer, 72 hrs culture; 6) Ea1C-35, roller bottle, 7-dayculture; 7) Ea1C-35 roller bottle/14-day culture; 8) Fa2N-4 monolayer/72hrs culture; 9) Fa2N-4 roller bottle/7-day culture; 10) Fa2N-4, rollerbottle/14-day culture.

FIG. 4 is a flowchart showing basic procedures for conducting enzymeinduction studies in primary cultures of human hepatocytes and Fa2N-4cells.

FIG. 5 is photomicrographs showing the morphology of human hepatocytes(left panel) and Fa2N-4 cells (right panel) at the light microscopylevel.

FIG. 6 is a set of graphs showing the induction of CYP enzymes byomeprazole and rifampin in Fa2N-4 cells (CYP1A2, CYP3A4, CYP2B6, andCYP2C9; DMSO control).

FIG. 7 is a graph depicting the reproducibility of CYP2B6 induction inrifampin-treated Fa2N-4 cells in 6-, 12- and 24-well plates.

FIG. 8 is a graph depicting the reproducibility of CYP1A2 and CYP3A4induction across multiple cell passages.

FIG. 9 is a summary graph depicting that the induction of CYP2B6 byrifampin is the same in 6-, 12- and 24-well plates.

FIG. 10 is a graph depicting the effect of cell culture format on theinduction of CYP1A2 by omeprazole and the induction of CYP3A4 byrifampin.

FIG. 11 is a graph showing the time course of CYP1A2 and CYP3A4induction in Fa2N-4 cells.

FIG. 12 is a graph showing the concentration-response curves for CYP1A2induction by omeprazole and for CYP3A4 induction by rifampin in Fa2N-4.

FIG. 13 is a graph showing that compounds shown previously to activatePXR and induce CYP3A4 in human hepatocytes induce CYP3A4 activity inFa2N-4 cells, whereas Ah receptor agonists do not.

FIG. 14 is a graph showing the range of CYP3A4 induction in primarycultures of human hepatocytes.

FIG. 15 is a graph showing the effect of enzyme inducers on CYP1A2 andCYP3A4 activity in Fa2N-4 cells (left panel, CYP1A2; right panel,CYP3A4).

FIG. 16 is a graph showing results of the use of the immortalizedhepatocytes in toxicity studies.

FIG. 17 is a phase contrast image of confluent Fa2N-4 cells plated in96-well Biocoat Type I collagen plates in MPE media at 200×magnification.

FIG. 18 is graphs depicting the induction of CYP1A2, CYP2C9, CYP3A4,UGT1A, and MDR1 transcripts in Fa2N-4 cells.

FIG. 19 is graphs showing the measurement of induction by cytochrome-450enzyme activity in Fa2N-4 cells ((A): Measurement of CYP3A4 activity;(B): Measurement of CYP2C9 activity; (C): Measurement of CYP1A2activity).

FIG. 20 is a graph showing EC50 plots for Fa2N-4 cells using increasedCYP3A4 transcript values (panel (A)) and increased CYP3A4 enzymeactivity (panel (B)).

FIG. 21 is a graph showing response of multiple passages of Fa2N-4 cellsto a CYP3A4 inducer with a weak response (50 μM dexamethasone) and aCYP3A4 inducer that exhibits a strong response (10 μM rifampin).

FIG. 22 is a graph showing induction of CYP3A4 transcript in Fa2N-4cells after 48 hour exposure to 10 μM rifampin (closed bars) is shown incomparison with vehicle (open bars).

FIG. 23 is an autoradiograph of a gel (gel 1) the lanes for which areshown in Table 9 in Example 10.

FIG. 24 is an autoradiograph of a gel (gel 2) the lanes for which areshown in Table 9 in Example 10.

FIG. 25 is an autoradiograph of a gel (gel 3) the lanes for which areshown in Table 9 in Example 10.

FIG. 26 is an autoradiograph of a gel (gel 4) the lanes for which areshown in Table 9 in Example 10.

FIG. 27 is an autoradiograph of a gel (gel 5) the lanes for which areshown in Table 10 in Example 10.

FIG. 28 is an autoradiograph of a gel (gel 6) the lanes for which areshown in Table 10 in Example 10.

FIG. 29 is an autoradiograph of a gel (gel 7) the lanes for which areshown in Table 10 in Example 10.

FIG. 30 is an autoradiograph of a gel (gel 8) the lanes for which areshown in Table 10 in Example 10.

FIG. 31 is a picture of gels after two-dimensional electrophoreticanalysis of the secreted proteins of the Fa2N-4 and Ea1C-35 cell lines((A): Fa2N-4; (B): Ea1C-35; (C): Western blot using anti-Factor IXantibody for Ea1C-35 to detect secreted Factor IX).

FIG. 32 is an autoradiograph of a gel (gel 8) the lanes for which areshown in Table 11 in Example 11.

FIG. 33 shows a photograph of an ELISA (plate 1) containing acolorimetric enzyme immunoassay for the quantitative determination ofsecreted hGH utilizing the sandwich ELISA principle; the key for thisplate is shown in Table 18.

FIG. 34 shows a photograph of an ELISA (plate 2) containing acolorimetric enzyme immunoassay for the quantitative determination ofsecreted hGH utilizing the sandwich ELISA principle; the key for thisplate is shown in Table 18.

FIG. 35 is a photograph of immunostained Fa2N-4 cells stained for CD81expression; CD81 was visualized by indirect immunofluorescence with afluorescein conjugated secondary antibody.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations and Terms

In accordance with the present invention and as used herein, thefollowing terms and abbreviations are defined with the followingmeanings, unless explicitly stated otherwise. These explanations areintended to be exemplary only. They are not intended to limit the termsas they are described or referred to throughout the specification.Rather, these explanations are meant to include any additional aspectsand/or examples of the terms as described and claimed herein.

The following abbreviations are used herein:

-   MCT=MultiCell Technologies-   MFE=Multi-functional Enhancing media-   TPP=therapeutic plasma proteins-   IαIp=inter-alpha-inhibitor proteins-   SV40=simian virus 40 T antigen and t antigen-   AAT=α-1-antitrypsin

The term “cell line” refers to a population or mixture of cells ofcommon origin growing together after several passages in vitro. Bygrowing together in the same medium and culture conditions, the cells ofthe cell line share the characteristics of generally similar growthrates, temperature, gas phase, nutritional and surface requirements. Thepresence of cells in the cell line expressing certain substances, forexample albumin, can be ascertained, provided a sufficient proportion,if not all, of the cells in the line produce a measurable quantity ofthe substance. An enriched cell line is one in which cells having acertain trait, e.g. expressing albumin, are present in greaterproportion after one or more subculture steps, than the original cellline.

The term “clonal cells” are those, which are descended from a singlecell. As a practical matter, it is difficult to obtain pure cloned cellcultures of mammalian cells. A high degree of cell purity can beobtained by successive rounds of cell enrichment. As used herein, a cellculture in which at least 80% of the cells possess a defined set oftraits is termed a cloned cell culture. Preferably, a cell culture inwhich at least 90% of the cells possess a defined set of traits istermed a cloned cell culture. More preferably, a cell culture in whichat least 98% of the cells possess a defined set of traits is termed acloned cell culture. The Fa2N-4 and Ea1C-35 cell lines claimed in thisinvention are clonal cell lines.

The term “immortalization” is defined as the acquisition of an infinitelife span. Immortalization may be induced in finite cell lines bytranfection with telomerase, oncogenes, or the large T antigen of theSV40, or by infection with SV40. Immortalization is not necessarily amalignant transformation, though it may be a component of malignanttransformation.

The term “immortalized” refers to the cell line that grows continuallywithout senescence when cultured in vitro in a suitable growth medium.

The term “virally-immortalized” refers to hepatocytes being transfectedor infected with all or part of the viral genome of a wild type ormutant virus. Preferably, the virus is a DNA virus. More preferably, thevirus is SV40, which binds to p53 and Rb tumor suppressor proteins,leading to inactivation of their tumor suppressor pathways.

The term “substantially pure” refers to a DNA which has been purifiedfrom the sequences which flank it in a naturally occurring state, i.e.,a DNA fragment which has been removed from the sequences which arenormally adjacent to the fragment, e.g., the sequences adjacent to thefragment in the genome in which it naturally occurs, and which has beensubstantially purified from other components which naturally accompanythe DNA, e.g., DNA which has been purified from the proteins whichnaturally accompany it in the cell.

The term “hepatocytes” refers to liver cells that are capable ofconsiderable regeneration in response to loss of liver mass (e.g.,through hepatotoxic processes, disease, or surgery), and constituteabout 80% of the cell population of the liver. They are large polygonalcells measuring between 20-30 μm. Hepatocytes have as many as 200-300peroxisomes per cells, which are involved in the breakdown of hydrogenperoxide, produced in many of the general cytoplasmic metabolicactivities. In addition, peroxisomes have specific oxidative functionsin gluconeogenesis, metabolism of purines, alcohol and lipids. Thesmooth endoplasmic reticulum (sER) in hepatocytes contain enzymesinvolved in degradation and conjugation of toxins and drugs. Underconditions of hepatocyte challenge by drugs, toxins or metabolicstimulants, the sER may become the predominant organelle in the cells.Hepatocytes perform multiple finely-tuned functions which are criticalto homeostasis. Of the variety of cell types in the mammalian body, onlyhepatocytes combine pathways for synthesis and breakdown ofcarbohydrates, lipids, amino acids, protein, nucleic acids andco-enzymes simultaneously to accomplish a unique biological task.

The term “isolated hepatocyte” refers to a hepatocyte that has beenphysically separated from other cells to which it is attached in itsnatural environment.

The term “primary hepatocyte” refers to a hepatocyte that has beenrecently isolated from intact liver tissue.

The term “normal primary human hepatocyte” refers to a hepatocytederived from a nondiseased human liver and maintained in vitro for afinite period when cultured in a suitable medium.

The term “cryopreserved human hepatocyte” refers to a normal primaryhuman hepatocyte that was cryopreserved prior to being cultured in asuitable medium.

The term “metabolic activity” refers to the sum total of the chemicalreactions that proceed in a cell, including catabolism (breaking down)and anabolism (building up). The metabolic activity in a hepatocyteincludes, but is not limited to, the ability to process potentiallytoxic compounds, e.g., a drug or endogenous metabolite, into a lesstoxic or non-toxic compound.

The term “cytochrome P450 enzyme” or “CYP” refers to a family ofheme-based oxidase enzymes found predominantly in the liver. Theseenzymes form the first line of defense against toxins and they areinvolved in the metabolism of hydrophobic drugs, carcinogens, and otherpotentially toxic compounds and metabolites circulating in the blood.They are found tethered to the surface of the endoplasmic reticulum,where they attach a chemical handle onto carbon-rich toxins. Then otherenzymes attach large soluble groups to these handles, making the entiremolecule more water soluble. This allows the toxins to be eliminated bythe urinary and digestive systems. The CYP family is divided intosubfamilies, which include, but are not limited to, CYP1A, CYP2A, CYP2C,CYP2D, CYP2E, and CYP3A. Within these subfamilies there are numeroushuman CYP enzymes, often termed “isozymes” or “isoforms.” The humanCYP3A, CYP2D6, CYP2C, and CYP1A isoforms are known to be important indrug metabolism. See, e.g., Murray, M., 23 Clin. Pharmacokinetics 132-46(1992). CYP3A4 is by far the major isoform in human liver and the smallintestines, comprising 30% and 70% respectively of the total CYP450protein in those tissues. Based primarily on in vitro studies, it hasbeen estimated that the metabolism of 40% to 50% of all drugs used inhumans involve CYP3A4 catalyzed oxidations. See Thummel, K. E. &Wilinson, G. R., In Vitro and In Vivo Drug Interactions Involving HumanCYP 3A, 38 Ann. Rev. Pharmacol. Toxicol., 389-430 (1998).

The term “hepatic function” refers to liver specific biologicalfunctions, which include, but are not limited to, (1) gluconeogenesis;(2) glycogen synthesis, storage, and breakdown; (3) synthesis of serumproteins including, but not limited to, albumin, hemopexin,ceruloplasmin, the blood clotting factors (including, but not limitedto, Factors V, VII, VIII, IX, X, prothrombin, and fibrinogen),α-1-antitrypsin, transferrin, and antithrombin III; (4) conjugation ofbile acids; (5) conversion of heme to bile pigments; (6) lipoproteinsynthesis; (7) vitamin storage and metabolism; (8) cholesterolsynthesis; (9) ammonia metabolism, including urea synthesis andglutamine synthesis; (10) amino acid metabolism, including metabolicconversion and re-utilization of aromatic amino acids; and (11)detoxification and drug metabolism.

Hepatocyte-derived proteins provide a safer, more reproducible approachfor producing native plasma proteins for therapeutic applications. Theseplasma proteins include Factor VIII, Factor IX, human growth hormone(hGH), and α-1-antitrypsin, but can also include other plasma proteins,such as growth factors. This finding is based upon MCT's data thatdemonstrates its proprietary, immortalized human hepatocyte cell lines,continue to produce inter-alpha-inhibitor proteins, a complex family ofplasma proteins made by four different polypeptides that are producedfrom four different genes. Salier, J.-P., et al., The inter-a-inhibitorfamily: from structure to regulation. Biochem J, 1996. 351: p. 1-9.

Methods of Use of Immortalized Cell to Produce Proteins

One aspect of the present invention is a method of use of immortalizedhuman hepatocyte cells to produce a protein, preferably, a therapeuticprotein, and more preferably a therapeutic plasma protein.

In general, such a method comprises the steps of:

(1) providing an immortalized human hepatocyte cell that includes DNAthat encodes and can express a protein;

(2) culturing the immortalized hepatocyte cell under conditions in whicha gene or genes encoding the protein are expressed so that the proteinis produced and processed in the immortalized hepatocyte cell; and

(3) isolating the processed protein from the immortalized hepatocytecell; wherein the protein is expressed such that the protein isprocessed and glycosylated, if necessary, so that its in vivo functionis substantially preserved after its isolation.

The protein to be produced can be a plasma protein, such as atherapeutic plasma protein. Examples of therapeutic plasma proteinsinclude, but are not limited, to, proteins such as Factor VIII, FactorIX, human growth hormone (hGH), α-1-antitrypsin, or a growth factor.Alternatively, the protein to be produced can be an IαIp proteincomplex, such as either IαI or PαI, or both of these protein complexes.In still another alternative, the protein can be a protein useful fordiagnostic uses or other uses such as albumin, transcobalamin II,C-reactive protein, fibronectin, or ceruloplasmin, as well as otherproteins having structural, enzymatic, or transport activities. In yetanother alternative, the protein to be produced can be a mutein of aprotein such as growth factors, blood clotting factors such as FactorVIII or Factor IX, human growth hormone, antitrypsins such asα-1-antitrypsin, or another protein whose primary structure is modifiedby standard techniques of genetic engineering, such as site-specificmutagenesis. Similarly, the protein can be a mutein of a protein usefulfor diagnostic uses or other uses such as albumin, transcobalamin II,C-reactive protein, fibronectin, or ceruloplasmin, as well as otherproteins having structural, enzymatic, or transport activities.

The protein to be produced can be a protein that is naturally producedby hepatocytes, either constitutively or in response to one or moreoutside stimuli, such as hormonal signals. Alternatively, the protein tobe produced can be a protein that is not naturally produced byhepatocytes. In the latter case, the protein can be a mutein of aprotein that is naturally produced by normal hepatocyte cells.

Many biologically-active proteins comprise at least two differentsubunits. When such a protein is expressed, the immortalized hepatocytecell can be transformed or transfected with at least two vectors, eachvector including: (1) DNA including at least one gene that encodes atleast one subunit of the protein; and at least one control elementoperably linked to the DNA encoding at least one gene that encodes thesubunit of the protein.

The immortalized liver cells can be the immortalized liver cellsdisclosed in Provisional Patent Application Ser. No. 10/510,509, by Liuet al., filed Oct. 10, 2003, and incorporated herein by this reference.In contrast to heterologous proteins produced by genetic recombinationin mammalian cells, such as Chinese Hamster Ovary cells, proteinsderived from immortalized liver cells as used in methods according tothe present invention behave more normally since the secondarypost-translational modifications required for complete function arecarried out by the hepatocytes directly. A significant advantage ofusing immortalized liver cells to produce proteins, preferablytherapeutic proteins, and more preferably therapeutic plasma proteins,is that the producer cell line is of human origin and therefore leads toa more natural protein. Therefore, since a number of therapeutic plasmaproteins (TPP) are synthesized by human hepatocytes, humanhepatocyte-based expression systems of the cell lines of the presentinvention are used to produce TPP in their “native” form. For one thing,this eliminates a possible immune response if a non-human protein weregiven to a human subject, as either a humoral or a cellular antibodyresponse could develop if a protein recognized as non-self wereadministered.

A large number of proprietary immortalized human hepatocyte cell linesare disclosed in Provisional Patent Application No. 60/510,509, by Liuet al., filed Oct. 10, 2003, and incorporated herein by this reference.The majority of these cell lines were created using simian virus 40(SV40) T antigen as the immortalization gene. This strategy was chosenbecause transfection of human cells with T antigen results in celllifespan extension and frequently in nontumorigenic immortalizationsince the cells are semipermissive to viral infection. T antigen is anuclear protein of 90,000 daltons. Cascio, S., Novel strategies forimmortalization of human hepatocytes. Artificial Orgs, 2001. 25: p.529-538.

The multiple mechanisms of T antigen action are still underinvestigation, but many studies demonstrate that this viral proteinbinds to and inactivates Rb and p53, two key tumor suppressor genes ofthe host cell. Ludlow, J., Interactions between SV40 large-tumor antigenand the growth suppressor proteins pRB and p53. FASEB J, 1993. 7: p.866-871.

While inactivation of Rb and p53 extends the lifespan of the cell,immortalization requires a secondary genetic event in order for the cellto escape senescence and proliferate indefinitely. The nature of thisevent is poorly understood, but occurs when the cells proceed throughcrisis. Most SV40 T antigen immortalized cell lines retain varyinglevels of the differentiated characteristics associated with the primarycell type and do not display tumorigenicity prior to extensive passagein vitro. Kuroki, T. and N. Huh, Why are human cells resistant tomalignant cell transformation in vitro? Jpn J Cancer Res, 1993. 84: p.1091-1100.

The normal human liver primary cells can be made to grow continuously bytransfecting the cells with the T antigen gene of SV40 virus.Transfection or infection can be accomplished by use of a virus or aplasmid containing the T antigen gene of the SV40 virus. Eithertransfection or infection may lead to transformation of the cell line.Other transformation vectors may also be useful, such as papilloma virusor Epstein Barr virus. The techniques for making continuous human celllines are described in the following references: Grahm. F. L., SmileyJ., Russell, W. C. and Nairn, R. Characteristics of a human cell linetransformed by DNA from human adenovirus type 5. J. Gen. Virol.,36:59-72 (1977); Zur Hausen, H. Oncogenic herpes viruses In: J. Tooze(ed.), DNA tumor viruses, Rev. Ed. 2, pp 747-798. Cold Spring Harbor,N.Y., Cold Spring Harbor Press (1981); Popovic, M., Lange-Wantein, G.,Sarin, P. S., Mann, D. and Gallo, R. C. Transformation of a humanumbilical cord blood T-cells by human T-cell leukemia/lymphoma virus(HTLV), Proc. Natl. Acad. Sci. USA, 80:5402-5406 (1983); DiPaolo, J. A.Pirisi, I., Popeseu, N. C., Yasumoto, S., Poniger, J. Progressivechanges induced in human and mouse cells by human Papillomavirus Type-16DNA, Cancer Cells 5:253-257 (1987).

Preferably, immortalized human hepatocytes useful in methods accordingto the present invention are derived from primary cryopreserved humanhepatocytes. Preferably, immortalized human hepatocytes useful inmethods according to the present invention are immortalized byintroduction of a substantially pure SV40 DNA. Preferably, thesubstantially pure SV40 DNA includes wild type SV40 large T and small tantigens (TAg). Typically, the hepatocytes include DNA from whichsubstantially pure DNA encoding a tumor suppressor gene can be isolated.Preferably, the tumor suppressor gene encoded by the DNA is human Rb.Preferably, the hepatocytes also include DNA from which substantiallypure DNA encoding human p53 can be isolated.

Typically, the virally-immortalized human hepatocyte has the ability tobe maintained and grow in serum-free media. Preferably, the serum-freemedia is MCT serum-free media.

Suitable hepatocyte cell lines include the virally-immortalized humanprimary hepatocyte cell line Fa2N-4, and the virally-immortalized humanhepatocyte cell line Ea1C-35. Other human hepatocyte cell lines can alsobe used.

Creation of Immortalized Human Hepatocyte Cell Lines

Primary Cell Isolation

Digestion of donor, human liver was performed in vitro withpre-perfusion of oxygen-saturated, calcium-free buffer at 37° C.Pre-perfusion continued until the liver was blanched and followed byperfusion with oxygen-saturated, collagenase buffer until the liver wasthoroughly digested (approximately 45 minutes).

To harvest cells, the liver was minced into 1 cm² pieces with theresulting suspension filtered through a #10 wire screen, then filteredagain through a 253 um nylon mesh. The suspension was centrifuged at20×g for five minutes at 4° C. to sediment intact parenchymal cells. Thepellet was resuspended at 4° C. and washed with washing buffer (3×) toremove all collegenase. The cell pellet was resuspended in 150 ml tissueculture media to yield a final volume of 400-500 ml with a density of3-4×10⁷ cells/ml. Trypan blue and lactate dehydrogenase viabilityassessment was performed on aliquots of this suspension.

Cryopreservation of Primary Human Hepatocytes

The freshly isolated human hepatocytes isolated from donor liver asdescribed above were washed with washing buffer three times bycentrifuging at 50×g for 5 minutes. The cell pellet was resuspended inchilled freezing medium (serum-free MFE medium: FBS:DMSO (8:1:1)) at afinal cell density of 5×10⁶/mL. Aliquots of the cell suspension weretransferred to Nunc Cryovials (1.0 mL/1.5 ml cryovial, 4.5 mL/5 mlcryovial). The cells in cryovials were equilibrated at 4° C. for 15-30minutes, the vials were then placed in a styrofoam container at −80° C.for at least 3 hours. The vials were then plunged in LN₂.

Creation of Cell Lines

Cryopreserved human hepatocytes were rapidly thawed in a 42 degreeCelsius water bath, washed and plated in MFE culture medium. Two dayslater the immortalizing gene was introduced by lipofection-mediatedtransfection. The Ea1C-35 cell line was derived from transfection withan immortalization vector containing the 2.5 kb early region of the SV40genome, which encompasses both the large-T and small-t antigens, andwhose expression is driven by the SV40 early promoter. This early regionwas inserted into the Stratagene pBluescript SK vector backbone and wasnamed pBlueTag. Neomycin resistance was conferred on the transfectedcells as a selectable marker by co-transfection of a neo plasmid. Cloneswere initially selected based on their ability to grow in G418containing media. The Ea1C-35 cell line was established and maintainedin CSM medium.

The Fa2N-4 cell line was immortalized via lipofection-mediatedtransfection with a single immortalization vector. The early region ofthe SV40 genome, contained in the pBlueTag vector, was inserted into abackbone based upon the InvivoGen pGT60mcs plasmid and was named pTag-1.The T-antigen coding region is under the influence of a hybrid hEF1-HTLVpromoter. The vector also encodes a hygromycin resistance gene as a drugselectable marker. Clones were selected based on their ability to growin hygro containing media. The Fa2N-4 cell line was established andmaintained in MFE.

The Fa2N-4 cell line was deposited under the terms of the BudapestTreaty at the American Type Culture Collection, 12301 Parklawn Dr.,Rockville, Md., on Oct. 6, 2003. The Ea1C-35 cell line was depositedunder the terms of the Budapest Treaty at the American Type CultureCollection, 12301 Parklawn Dr., Rockville, Md., on Oct. 6, 2003.

Suitable immortalized hepatocyte cell lines, such as those describedabove, can be used for the production of proteins as described above.The proteins can be therapeutic plasma proteins such as Factor VIII,Factor IX, α-1-antitrypsin, human growth hormone, growth factors, orother proteins. The proteins can also be muteins of proteins such asgrowth factors, blood clotting factors, antitrypsins such asα-1-antitrypsin, and other proteins whose primary structure is modifiedby standard techniques of genetic engineering, such as site-specificmutagenesis. The proteins can further also include other proteins oftherapeutic or diagnostic interest including albumin, transcobalamin II,C-reactive protein, fibronectin, or ceruloplasmin, as well as otherproteins having structural, enzymatic, or transport activities.

When the hepatocyte cell line is used for the production of anaturally-occurring plasma protein, such as, but not limited to, FactorVIII, Factor IX, human growth hormone (hGH), or α-1-antitrypsin, severalstrategies can be used to maximize expression of the plasma protein.

In one strategy, expression of the naturally-occurring gene encoding theprotein is enhanced by introduction of a high-level promoter. Suchpromoters are known in the art, and are described, for example, in S. B.Primrose et al., “Principles of Gene Manipulation” (6^(th) ed., 2001,Blackwell, Oxford, England), p. 199, incorporated herein by thisreference. Such promoters can also be accompanied by additional controlelements, such as enhancers. Such promoters or promoter-enhancercombinations include the SV40 early promoter and enhancer, the Roussarcoma virus long-terminal-repeat promoter and enhancer, and the humancytomegalovirus immediate early promoter.

However, it is generally preferred to enhance expression by introducingmultiple copies of the gene encoding the protein to be expressed, asubunit of the protein to be expressed, or a precursor of the protein tobe expressed via the use of one or more recombinant vectors thatinclude: (1) the gene encoding the protein to be expressed, a subunit ofthe protein to be expressed, or a precursor of the protein to beexpressed; and (2) at least one control element affecting thetranscription of the gene, the control element being operably linked tothe gene. The control element is typically a promoter or apromoter-enhancer combination. The characteristics of a suitable vectoralso include: (1) an origin of replication; (2) restriction endonucleasecleavage sites allowing the insertion of DNA encoding the desired genes;and (3) a selection marker, typically one that confers antibioticresistance. In one particularly preferred embodiment, the controlelements comprise at least one promoter and at least one enhancer.

Suitable recombinant vectors include, but are not limited to,SV40-derived vectors, murine polyoma-derived vectors, BK virus-derivedvectors, Epstein-Barr virus-derived vectors, adenovirus-derived vectors,adeno-associated virus-derived vectors, baculovirus-derived vectors,herpesvirus-derived vectors, lentiviral-derived vectors,retrovirus-derived vectors, alphavirus-derived vectors, vacciniavirus-derived vectors, and others. Such vectors typically include astrong and constitutive promoter, at least one intron in the DNA to beexpressed, and a polyadenylation signal at the 3′-terminus of thesequence to be transcribed. The addition of a signal peptide to ensureappropriate post-translational modification, such as glycosylation, canbe desirable. These vectors and characteristics of vectors are describedin S. B. Primrose et al., “Principles of Gene Manipulation” (6^(th) ed.,2001, Blackwell, Oxford, England), pp. 174-201, and in T. A. Brown,“Gene Cloning and DNA Analysis: An Introduction” (4^(th) ed., 2001,Blackwell, Oxford, England), both of which are incorporated herein bythis reference.

Methods for isolating DNA encoding proteins to be expressed and forinserting such DNA into these vectors are also well known in the art.These methods are described, for example, in S. B. Primrose, “Principlesof Gene Manipulation” (6^(th) ed., Blackwell, Oxford, 2001),incorporated herein by this reference. In general, suitable DNA forcloning can be obtained from reverse transcription of specific mRNAs,which can be followed by application of the polymerase chain reaction(PCR) to amplify the DNA; such DNAs are generally known as cDNA. DNA canbe inserted into the vectors by techniques that generally involvecleavage of the vectors with specific restriction endonucleases andinsertion of the DNA at the cleavage sites.

Methods for transforming or transfecting the virally-immortalized humanhepatocytes are well-known in the art and need not be described furtherin detail here. In general, such methods include, but are not limitedto, lipofection, calcium-phosphate-mediated transfection, transfectionmediated by DEAE-dextran, transfection by electroporation, transfectionby biolistics, and transfection using polybrene. These transfectionmethods are described in J. Sambrook & D. W. Russell, “MolecularCloning: A Laboratory Manual (3d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York, 2001), vol. 3, ch. 16, incorporatedherein by this reference.

In many cases, it is desirable to incorporate one or more reporter genesinto the vector to assess the efficiency of transfection. The gene ofchoice is under the control of strong ubiquitous promoter-enhancercombinations. These include those from the immediate early genes ofhuman cytomegalovirus, the Rous sarcoma virus long terminal repeat, orthe human β-actin gene. An example of a suitable reporter gene is thechloramphenicol acetyltransferase (CAT) gene found in the Escherichiacoli transposon. Detection of expression of the reporter gene can bedone by a variety of techniques, such as detection of fluorescence ordetection of radioactive products. Reporter genes and their assay arefurther described in M. A. Aitken et al., “Gene Transfer and Expressionin Tissue Culture Cells of Higher Eukaryotes,” in Molecular BiomethodsHandbook (R. Rapley & J. M. Walker, ed., Humana Press, Totowa, N.J.,1998), pp. 235-250, incorporated herein by this reference.

Once the protein has been expressed, it is then necessary to isolate theexpressed protein. This is typically performed by standard methods forprotein purification. These methods include, but are not limited to,precipitation with salts such as ammonium sulfate, ion-exchangechromatography, gel filtration chromatography, reverse phase highpressure liquid chromatography, electrofocusing, chromatofocusing,and/or immunoaffinity chromatography, using any readily ascertainableproperty, such as protease activity, to detect the protein. Otherpurification methods are also known in the art. Protein purificationmethods are described, for example, in R. K. Scopes, “ProteinPurification: Principles and Practice” (3d ed., Springer-Verlag, NewYork, 1994), incorporated herein by this reference.

In some cases, the expressed protein can be secreted from the cell intothe surrounding culture medium. The efficiency of this process dependson the pattern of post-transcriptional modification, such asglycosylation, that the protein undergoes. This pattern affects theprocessing of the protein within the rough endoplasmic reticulum and theGolgi apparatus and its subsequent secretion. This is described in A. J.Dorner & R. J. Kaufman, “Analysis of Synthesis, Processing, andSecretion of Proteins Expressed in Mammalian Cells” in Gene ExpressionTechnology (D. V. Goeddel, ed., Academic Press, San Diego, 1991), pp.577-598, incorporated herein by this reference. The cloning vector canalso be chosen so that the protein being expressed is fused to anotherprotein, called a tag, which can be used to facilitate proteinpurification. Examples of tags include glutathione S-transferase, theMalE maltose-binding protein, and a polyhistidine sequence. Theresulting fusion protein can then be cleaved with specific proteolysisto remove the tag and result in purified plasma protein. This techniquecan be applied to both therapeutic and non-therapeutic proteins,including plasma proteins.

In one example of the application of the present invention to theproduction of proteins, such virally-immortalized primary humanhepatocytes can be transformed or transfected with vectors that includethe genes for the various precursors for the IαIp proteins, such as IαIand PαI. This is done to increase the expression of the IαIp proteins.In one version of this example, two vectors are used: (1) a first vectorthat includes the genes ITH3 and AMBP; and (2) a second vector thatincludes the genes ITH2 and ITH1.

In one example of the production of therapeutic plasma proteins, thevector that includes DNA encoding the ITH1 and ITH2 genes is a variantof a commercially-available vector sold by InvivoGen. The vector sold byInvivoGen is designated pVITRO2 and contains a hygromycin resistancegene. The variant vector is designated pVITRO2-Blasti. The vector ismodified by replacing the hygromycin resistance gene with a blasticidinresistance gene. The vector uses two human ferritin composite promoters.The 5′-UTR of FerH and FerL are replaced by the 5′-UTR of the mouse andchimpanzee EF1a genes. The activity of both promoters is increased bythe addition of the SV40 and CMV enhancers to yield activity similar tothat of the CMV promoter. This promoter backbone was chosen in orderthat similar levels of expression for the two cDNAs that have beenincorporated are driven from each vector. This is particularly desirablebecause the IαIp proteins are built in a 1:1:1:1 ratio using thepolypeptides encoded by the cDNAs. In the plasmid pVITRO2-Blasti, ITH1is cloned into MCS1 and ITH2 into MCS2. The cDNAs have uniquerestriction sites added to their ends to facilitate possible downstreamsubcloning. The linear schema for the cDNA encoding ITH1 is5′-BamHI-AgeI-EcoRV-cDNA-MluI-AvrII-BamHI-3′. The linear schema for thecDNA encoding ITH2 is 5′-FspI-SgrA1-cDNA-XhoI-FspI-3′.

Similarly, in one example of the production of therapeutic plasmaproteins, the vector that includes DNA encoding the AMBP and ITH3 genesis another variant of the commercially-available pVITRO2 vector. Thisvariant is designated pVITRO2-Neo and contains a neomycin resistancegene in place of the hygromycin resistance gene. This vector uses thesame promoter backbone and 5′-UTRs as pVITRO2-Blasti. This vectorcontains the DNA encoding AMBP in MCS1 and the DNA encoding ITH3 inMCS2. The linear schema for the cDNA encoding AMBP is5′-BamHI-AgeI-EcoRV-cDNA-MluI-AvrII-BamHI-3′. The linear schema for thecDNA encoding ITH3 is 5′-XhoI-NheI-cDNA-BglII-XhoI-3′. Other vectors canbe used, but it is generally preferred that the vectors express each ofthe encoded proteins at approximately equal rates because of the 1:1ratios between protein subunits that exist in the protein complexes.

Accordingly, another example of the application of the present inventionin producing plasma proteins is a vector that includes DNA encoding thegenes ITH3 and AMBP operably linked to at least one control sequencecompatible with a suitable host cell. The control sequence can be apromoter, enhancer, or other control sequence.

Yet another example of the application of the present invention inproducing plasma proteins of the present invention is a vector thatincludes DNA encoding the genes ITH2 and ITH1 operably linked to atleast one control sequence compatible with a suitable host cell. Thecontrol sequence can be a promoter, enhancer, or other control sequence.

Yet another example of the application of the present invention inproducing therapeutic plasma proteins is an isolated and purifiednucleic acid sequence that includes nucleic acid encoding the genes ITH3and AMBP operably linked to at least one control sequence compatiblewith a suitable host cell. The control sequence can be a promoter,enhancer, or other control sequence. The nucleic acid sequence istypically DNA, but can be RNA or an RNA-DNA hybrid.

Similarly, yet another example of the application of the presentinvention in producing therapeutic plasma proteins is an isolated andpurified nucleic acid sequence that includes nucleic acid encoding thegenes ITH2 and ITH1 operably linked to at least one control sequencecompatible with a suitable host cell. The control sequence can be apromoter, enhancer, or other control sequence. The nucleic acid sequenceis typically DNA, but can be RNA or an RNA-DNA hybrid.

Therefore, as a result of the existence of these vectors, another aspectof the invention is a virally immortalized human hepatocyte that istransformed or transfected with at least one vector that includes: (1)DNA including at least one gene encoding a plasma protein; and (2) atleast one control element operably linked to the DNA encoding the plasmaprotein in order to enhance expression of the plasma protein. Thisvirally immortalized human hepatocyte therefore encodes DNA that encodesand can express a plasma protein. An example of this is a virallyimmortalized human hepatocyte that is transformed or transfected with atleast one vector that includes: (1) DNA including at least one gene fora precursor of a protein that is part of an IαIp protein complex; and(2) at least one control element operably linked to the DNA encoding atleast one precursor gene in order to enhance expression of the precursorgene.

Yet another aspect of the invention is transformed or transfectedeukaryotic cells, other than human hepatocyte cells, that produce activeIαIp, either IαI or PαI. In general, these cells are transformed ortransfected with at least one vector that includes: (1) DNA including atleast one gene for a precursor of a protein that is part of an IαIpprotein complex; and (2) at least one control element operably linked tothe DNA encoding at least one precursor gene in order to enhanceexpression of the precursor gene. These cells can be CHO cells, COScells, yeast cells, or other eukaryotic cells that can be transformed ortransfected, preferably with the vectors described above. These cellsproduce either IαI or PαI, or both; they also may produce otherproteins. These cells, therefore, are transformed or transfected witheither: (1) DNA encoding the genes ITH3 and AMBP; (2) DNA encoding thegenes ITH2 and ITH1; or (3) both DNA encoding the genes ITH3 and AMBPand DNA encoding the genes ITH2 and ITH1.

Yet another example of the production of therapeutic plasma proteinsaccording to the present invention is a method of using thesetransformed or transfected eukaryotic cells, other than human hepatocytecells, to produce IαIp.

In general, such a method comprises the steps of:

(1) providing a transformed or transfected eukaryotic cell, other than ahuman hepatocyte cell, that includes DNA that encodes and can expressproteins forming an IαIp protein complex;

(2) culturing the immortalized eukaryotic cell under conditions in whichgenes encoding proteins forming an IαIp protein complex are expressed sothat an IαIp complex is produced; and

(3) isolating the expressed IαIp protein complex from the immortalizedeukaryotic cell.

The protein produced can be either IαI or PαI, or both of these proteincomplexes.

Isolation of such protein complexes from cells expressing them, eithervirally immortalized human hepatocytes or cells other than humanhepatocytes, is performed by standard methods for protein purificationas described above.

Accordingly, another aspect of the present invention is the use of aprotein produced by the immortalized hepatocyte cells to treat a diseaseor condition. The disease or condition can be a disease or conditionaffecting the liver, such as sepsis, liver cancer, hepatitis, or liverfailure, or can be a disease or condition affecting an organ other thanthe liver, such as cancer at sites other than the liver, jointinflammation, or arthritis. An example of such a protein is an IαIpprotein complex, but the method is applicable to proteins in general.

An example of the use of therapeutic plasma proteins according to theinvention is the use of an IαIp protein complex.

In general, such a method comprises the steps of:

(1) providing an active IαIp protein complex; and

(2) administering the active IαIp protein complex to a patient sufferingfrom a disease or condition in a therapeutically active quantity totreat the disease or condition.

As stated above, the disease or condition can affect the liver, such assepsis, cancer, hepatitis, or liver failure. Alternatively, the diseaseor condition can affect an organ other than the liver, such as cancer atsites other than the liver, joint inflammation, or arthritis. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g. A.S. Nies & S. P. Spielberg, “Principles of Therapeutics” in J. G. Hardman& L. E. Limbird, eds., “Goodman & Gilman's The Pharmacological Basis ofTherapeutics” (9^(th) ed., McGraw-Hill, New York, 1996), ch. 3., pp.43-62.) It should be noted that the attending physician would know howto and when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministered dose of an IαIp protein complex in the management of aliver disease or condition will vary with the severity of the disease orcondition and with the route of administration, Further, the dose andperhaps dose frequency, will also vary according to the age, bodyweight, and response of the individual patient, as well as otherconditions affecting pharmacodynamic parameters such as liver and kidneyfunction. In general, however, for therapeutic protein compositions,routes other than oral are typically preferred in order to avoid theproteolytic activity of the digestive tract. These routes can beintravenous, intramuscular, intraperitoneal, intralymphatic,subcutaneous, or other routes.

As described above, the active IαIp protein complex can be produced inimmortalized hepatocytes, or can be produced in other eukaryotic cells.

Yet another aspect of the present invention is pharmaceuticalcompositions for treating a disease or condition comprising an activeprotein produced by eukaryotic cells, either virally immortalized humancells or other eukaryotic cells, in a quantity that is therapeuticallyeffective to treat the disease and a pharmaceutically acceptablecarrier. The pharmaceutical compositions of the invention comprising theactive protein can be in a variety of dosage forms which include, butare not limited to, solid, semi-solid and liquid dosage forms such astablets, pills, powders, liquid solutions or suspensions, suppositories,polymeric microcapsules or microvesicles, liposomes, and injectable orinfusible solutions. The preferred form depends upon the mode ofadministration and the particular therapeutic application.

Conventional pharmaceutically acceptable carriers for the compositionsmay include those known in the art such as serum proteins includinghuman serum albumin, buffer substances such as phosphates, water orsalts or electrolytes.

Yet another aspect of the present invention is antibodies thatspecifically bind protein produced according to the methods of thepresent invention. The protein can be used to prepare antibodies,including polyclonal antibodies that bind to purified protein orpeptides isolated from the protein. These antibodies can be used topurify the protein in larger quantities. For example, the protein can bepurified by fixing the antibody to a solid support and reacting theantibody fixed to the solid support with a sample containing the proteinto bind the protein to the antibody. Alternatively, the antibody can belabeled with a detectable label, reacting the antibody labeled with thedetectable label with a sample containing protein to bind the protein tothe antibody, thereby forming an antigen-antibody complex, andseparating the antigen-antibody complex from other proteins present inthe sample. In either case, the protein can then be dissociated from theantibody by standard techniques, such as high salt, change of pH, or lowconcentrations of chaotropic agents.

The purified protein complex can then be used, for therapeutic orscreening uses.

Monoclonal antibodies reactive with vesicle membrane transport proteincan be produced by hybridomas prepared using known procedures, such asthose introduced by Kohler and Milstein (see Kohler and Milstein,Nature, 256:495-97 (1975)), and modifications thereof, to regulatecellular interactions.

These techniques involve the use of an animal which is primed to producea particular antibody. The animal can be primed by injection of animmunogen (e.g. the IαIp protein complex) to elicit the desired immuneresponse, i.e. production of antibodies from the primed animal.Lymphocytes derived from the lymph nodes, spleens or peripheral blood ofprimed (immunized) animals can be used to search for a particularantibody. The lymphocyte chromosomes encoding desired immunoglobulinsare immortalized by fusing the lymphocytes with myeloma cells, generallyin the presence of a fusing agent such as polyethylene glycol (PEG). Anyof a number of myeloma cell lines can be used as a fusion partneraccording to standard techniques; for example, the P3-NS1/1-Ag4-1,P3-x63-Ag8.653, Sp2/0-Ag14, or HL1-653 myeloma lines. These myelomalines are available from the ATCC, Rockville, Md. Other myeloma linescan be used.

The resulting cells, which include the desired hybridomas, are thengrown in a selective medium such as HAT medium, in which unfusedparental myeloma or lymphocyte cells eventually die. Only the hybridomacells survive and can be grown under limiting dilution conditions toobtain isolated clones. The supernatants of the hybridomas are screenedfor the presence of the desired specificity, e.g. by immunoassaytechniques using the protein that have been used for immunization.Positive clones can then be subcloned under limiting dilutionconditions, and the monoclonal antibody produced can be isolated.

Both polyclonal and monoclonal antibodies can be obtained that arespecific for the protein of interest.

Various conventional methods can be used for isolation and purificationof the monoclonal antibodies so as to obtain them free from otherproteins and contaminants. Commonly used methods for purifyingmonoclonal antibodies include ammonium sulfate precipitation, ionexchange chromatography, arid affinity chromatography (see Zola et al.,in Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurell(ed.) pp. 51-52 (CRC Press, 1982)). Hybridomas produced according tothese methods can be propagated in vitro or in vivo (in ascites fluid)using techniques known in the art (see generally Fink et al., Prog.Clin. Pathol., 9:121-33 (1984), FIG. 6-1 at p. 123).

Generally, the individual cell line can be propagated in vitro, forexample, in laboratory culture vessels, and the culture mediumcontaining high concentrations of a single specific monoclonal antibodycan be harvested by decantation, filtration, or centrifugation.

In addition, fragments of these antibodies containing the active bindingregion reactive with the protein, such as Fab, F(ab′)₂ and F_(v)fragments can be produced. Such fragments can be produced usingtechniques well established in the art (see e.g. Rousseaux et al., inMethods Enzymol., 121:663-69, Academic Press (1986)).

Yet another aspect of the present invention is a screening method forthe detection of a protein for the detection of disease. As diseasedevelops, there is a drop in the expression of active protein. Ingeneral, one embodiment of a screening method according to the presentinvention comprises:

(1) providing a plasma sample from a patient;

(2) determining the concentration of an active protein in the plasmasample; and

(3) correlating the concentration of the active protein in the plasmasample with the presence or absence of disease in the patient todetermine the presence or absence of disease in the patient.

Typically, the concentration of active protein in the plasma sample isdetermined by an immunoassay using the antibodies described above. Anumber of immunoassay formats can be used, such as ELISA,radioimmunoassay, and other formats well known in the art. Eithercompetitive or non-competitive immunoassays can be used. Typically, theantibody is labeled, but it is also possible to label the antigen, suchas for a competitive immunoassay. Various types of labels can be used,including radioactive labels, fluorescent labels, chemiluminescentlabels, bioluminescent labels, and enzyme labels. Immunoassays aredescribed in E. Harlow & D. Lane, “Antibodies: A Laboratory Manual”(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988),ch. 14, pp. 553-612, incorporated herein by this reference.

In an alternative screening method, the quantity of mRNA encoding theprotein is detected, as this is related directly to expression. In oneembodiment of this alternative, the screening method comprises:

(1) taking a sample of cells from a patient;

(2) isolating the mRNA from the sample;

(3) determining the quantity or concentration of mRNA in the sampleencoding the protein; and

(4) correlating the quantity or concentration of mRNA in the sampleencoding the protein with the presence or absence of disease in thepatient to determine the presence or absence of disease in the patient.

Methods for isolation of mRNA from cells and tissues are well known inthe art and need not be described further in detail here. Such methodsare described in detail in J. Sambrook & D. W. Russell, “MolecularCloning: A Laboratory Manual” (3d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001), vol. 1, ch. 7, incorporatedherein by this reference. One method for performing the step ofdetermining the quantity or concentration of mRNA in the sample encodingthe IαIp protein complex is to perform the polymerase chain reaction(PCR) using an oligo-dT primer, with reverse transcriptase, to make aDNA copy of the polyadenylated mRNA in the sample. The DNA of the copyis then amplified in a second PCR reaction using primers specific forthe IαIp protein complex genes. This approach is described in C. R.Cantor & C. L. Smith, “Genomics: The Science and Technology Behind theHuman Genome Project” (John Wiley & Sons, 1999), pp. 119-120,incorporated herein by this reference. Another method for performing thestep of determining the quantity or concentration of mRNA in the sampleencoding the IαIp protein complex is the use of Northern blotting todetect specific mRNAs by hybridization. This requires that a suitableDNA labeled probe be available; typically, the labeled probe isradioactively labeled. This method is well known in the art and isdescribed, for example, in P. A. Sabelli, “Northern Blotting” inMolecular Biomethods Handbook (R. Rapley & J. M. Walker, eds., HumanaPress, Totowa, N.J., 1998), ch. 9, pp. 89-94, incorporated herein bythis reference.

Post-translational modifications of therapeutic proteins may affectbioactivity, clearance rate in vivo, immunogenicity and/or stability.Proteins secreted by our hepatocyte-based expression systems of thepresent invention behave more naturally than recombinant counterparts.For example, the inventors have demonstrated that its immortalized humanhepatocyte cell lines produce biologically active IαIp and therefore isa strong commercial source for this protein that cannot be produced byrecombinant technology. Therefore, the inventors' production of IαIp inits “native” form leads to a more effective, safe, and cost effectivesolution to treating life threatening diseases such as sepsis.

The use of cell lines suitable for the production of protein also allowsthe use of a sequential protein purification scheme that generatesmultiple products similar to plasma-derived proteins without thereoccurring risk of viral contamination. These hepatocyte-derived plasmaproteins provide a safe, effective, and cost efficient strategy tocommercially produce native plasma proteins, which overcomes theshortcomings of the prior art.

Utility

Examples of Uses of Virally Immortalized Liver Cell Lines That ProducePlasma Protein Complexes

The following are uses of virally immortalized liver cell lines thatproduce plasma proteins, more particularly, IαIp protein complexes.These uses are related to their production of IαIp protein complexes, aswell as to their possible production of other proteins. (1)Identification of potential chemotherapeutic drugs: These cells areuseful for screening chemicals suitable for the treatment of cancer andrelated diseases, by growing them in vitro in medium containing thechemical to be tested and then, after a suitable period of exposure,determining whether and to what extent cytotoxicity has occurred, e.g.by trypan blue exclusion assay or related assays (Paterson, MethodsEnzymol, 58:141 (1979)), or by growth assays such as colony formingefficiency (MacDonald et al, Exp. Cell. Res., 50:417 (1968)), all ofwhich are standard techniques well known in the art. (2) Investigationof the controls of gene expression by biological agents that induce orinhibit gene expression. Chemical and biological substances are screenedfor their ability to induce or inhibit gene expression or metabolicpathways by adding them to the growth medium of these liver cells andthen after a suitable period of time, determine whether a complex ofchanges, including cessation of DNA synthesis, induction or inhibitionof gene expression (as measured by RT-PCR analysis) and production ofliver specific proteins (as determined by immunochemical techniques)occurs. Identification of the effects of chemical and biologicalsubstances on the induction or inhibition of gene expression andmetabolic pathways is a way to identify new drug targets for treatingdiseases such as cancer.

(3) Studies of metabolism of carcinogens and other xenobiotics:Carcinogens and other xenobiotics may be added to the growth medium ofthese cells and the appearance of metabolic products of these compoundsmay be monitored by techniques such as thin layer chromatography or highperformance liquid chromatography and the like, and the interaction ofthe compounds and/or their metabolites with DNA is determined.

(4) Studies of DNA mutagenesis: Substances known or suspected to bemutagens may be added to the growth medium of the cells and thenmutations may be assayed, e.g., by detection of the appearance of drugresistant mutant cell colonies (Thompson, Methods Enzymol, 58:308,1979).

(5) Studies of malignant transformation by chemical, physical and viralagents, and transferred genes including oncogenes and high molecularweight genomic DNA from tumors, using standard assays such as anchorageindependent growth or tumor formation in athymic nude mice. For example,a cloned viral oncogene N-ras (an oncogene present in many liver cellcancers) can be introduced into the hepatocyte cells using strontiumphosphate transfection. The subsequent ability of the newly transfectedcells to form tumors in mice as well as grow in an anchorage-independentfashion can be assessed.

(6) Use of cells altered by transfer of oncogenes as in paragraph (5)above to screen for potential chemotherapeutic agents (by the techniquesdescribed in paragraph (1) above) especially those which may be specificfor cells transformed by the activation of particular oncogenes orcombination of oncogenes.

(7) Studies of cellular biochemistry, including changes in intracellularpH and calcium levels, as correlated with cell growth and action ofexogenous agents including but not limited to those described inparagraphs (1) through (6) above. To study intracellular pH and calciumlevels, cells in suitable culture vessels are exposed to fluorescentindicator dyes and then fluorescence emissions are detected with afluorescence spectrophotometer (Grynkiewicz et al, J. Biol. Chem.,260:3440-3450 (1985)).

(8) Studies of cellular responses to growth factors and production ofgrowth factors: Identification and purification of growth factorsimportant for growth and differentiation of human liver hepatocytecells. These cells are particularly useful for such an application sincethey grow in serum-free media. Therefore, responses to growth factorscan be studied in precisely defined growth medium and any factorsproduced by the cells may be identified and purified without thecomplication of the presence of serum.

(9) Use of recombinant DNA expression vectors to produce proteins ofinterest. This is described above for IαIp protein complexes; similartechniques can be used for other proteins. For example, the geneencoding a protein of therapeutic value may be recombined withcontrolling DNA segments (i.e. containing a promoter with or without anenhancer sequence), transferred into the cell (e.g., by strontiumphosphate transfection) and then the protein produced may be harvestedfrom the culture supernatant or a cellular extract by routine procedureswell known in the art.

(10) Studies of intracellular communication e.g., by dye scrape loadingassays, to determine whether the cells growing in vitro have the abilityto communicate via gap junctions. The cultures may be scraped, e.g.,with a scalpel, in the presence of a fluorescent dye in the growthmedium. Cells at the edge of the wound are mechanically disrupted andtherefore take up dye; whether intercellular communication has occurredmay be ascertained by determining whether cells distant from the woundalso contain dye.

(11) Characterization of cell surface antigens: The cells are incubatedwith an antibody against the cell surface antigen of interest, and thenreacted with a second antibody, which is conjugated to a fluorescentdye. The cells are then evaluated using a fluorescence activated cellsorter to determine whether they are fluorescent and therefore possesthe cell surface antigen.

(12) Cell-cell hybrid studies for identification of tumor suppressoractivity (Stranbridge et al, Science, 215:252-259 (1982)). To determinewhether these cell lines contain tumor suppressor genes, they are fusedto malignant tumor cells. The presence of tumor suppressor genes isindicated by loss of malignancy e.g., as detected by loss of ability toform tumors in athymic nude mice, in the hybrid cells.

(13) Identification of novel genes, including transforming genes in thenaturally occurring cancer described in paragraph (5) above, growthfactor genes as described in paragraph (8) above, tumor suppressor genesas described in paragraph (12) above, using standard molecularbiological techniques (Davis et al, Methods in Molecular Biology, NewYork: Elsevier (1986)) and techniques such as cDNA subtraction cloningand similar processes.

(14) Growth of replicating hepatitis virus (as e.g., HBV, non-A non-B,HAV and other livertropic virus, e.g., CMV). Establishment of a clonalcell line of human liver hepatocyte cells containing replicatinghepatitis virus using methods of transfection established for humanliver cancer cell lines (Sells, M. A. et al, Proc. Natl. Acad. Sci.,84:444-448). Using human liver hepatocyte lines, which contain HBV, theability of HBV, alone as well as in conjunction with chemical livercarcinogens such as aflatoxin B, can be evaluated for malignanttransformation using anchorage independent growth assays as well asgrowth in athymic nude mice. Cell-cell hybrid techniques similar tothose in paragraph

(13) can be used to evaluate possible inactivation of tumor suppressorgenes by fusion with malignant cells before and after HBV transfection.

The screening kits are easily assembled as other screening kitscontaining cell lines with other conventional components and labelinginstructions for performing the test.

(15) The immortalized cells may be used as a way of expanding cells forliver transplant and liver function assist devices, both implanted andextracorporeal. Also, these cells can have additional genestransfected/infected into them for organ transplant for therapy ofinherited metabolic disorders, especially those diseases associated withhepatic degradation (i.e., certain diseases are due to a deletion orabnormality of a particular gene). This gene could then be transfectedinto cells useful for production of IαIp protein complexes, and thecells then expanded for organ transplant.

(16) Studies of cytotoxicity of drugs, carcinogens, xenobiotics: Drugs,carcinogens, xenobiotics may be added to the growth medium of the cellsand the viability of the cells as a function of time of exposure may beascertained using gene expression profiling, dye exclusion, enzymeleakage, colony forming efficiency, etc. assays.

(17) Studies of gene expression: Drugs, chemicals, new chemicalentities, etc., may be added to the culture medium of the cells andchanges in gene expression as a function of exposure may be monitoredusing RNA and protein expression as biological endpoints. Changes mayreflect either induction or inhibition of specific genes. For example,cells may be cultured with drugs, chemicals, new chemical entities, etcto identify those agents that modulate the expression of drug metabolismenzymes including but not limited to cytochrome P450s designated CYP3A4or CYP1A2, the multi drug resistance gene, biliary transporters,glucuronyl transferases, glutathione transferases, sulfatases, etc.

(18) Studies of liver parasites: The cultured cells could proveefficacious for studying the life cycle of parasites that invadehepatocytes.

(19) Production of hepatocyte-derived proteins. This is describedextensively above with respect to the expression and production ofplasma proteins, particularly therapeutic plasma proteins, by thesecells. Cells maintained in suitable medium will naturally expressproteins such as blood clotting factors (e.g. Factor VIII and FactorIX), α-1-antitrypsin, human growth hormone, growth factors, etc., thatmay be purified and used. These proteins can be any proteins that areexpressible by differentiated human hepatocytes. This category ofproteins includes proteins that are naturally encoded and expressed bythose cells, either constitutively or in response to one or more outsidestimuli, such as hormonal signals. This category of proteins alsoincludes proteins that can be expressed by those cells in such a waythat they are processed and glycosylated so that their in vivo functionis substantially preserved when genes for those proteins are introducedinto those cells. This can include muteins of proteins such as growthfactors, blood clotting factors, antitrypsins such as α-1-antitrypsin,and other proteins whose primary structure is modified by standardtechniques of genetic engineering, such as site-specific mutagenesis.This can also include other proteins of therapeutic or diagnosticinterest including albumin, transcobalamin II, C-reactive protein,fibronectin, or ceruloplasmin, as well as other proteins havingstructural, enzymatic, or transport activities.

Proteins produced by methods according to the present invention can beused to treat a large variety of conditions, both conditions affectingthe liver and conditions affecting organs other than the liver. Thelatter can include cancer at sites other than the liver, jointinflammation, and arthritis. These proteins can include IαIp complexesand other proteins produced by these methods, as described above.

The properties of the cell lines provide the ability to performadditional screening assays of value.

For example, the invention includes a method of screening a compound forchemotherapeutic activity comprising the steps of:

(1) providing a hepatocyte cell line selected from the group consistingof Fa2N-4 and Ea1C-35;

(2) exposing the cell line to the compound to be screened; and

(3) determining the extent of cytotoxicity induced by the compound to bescreened in the cell line to determine whether the compound haschemotherapeutic activity.

The invention also includes a method of screening a compound formutagenic activity comprising the steps of:

(1) providing a hepatocyte cell line selected from the group consistingof Fa2N-4 and Ea1C-35;

(2) exposing the cell line to the compound to be screened; and

(3) determining the extent of mutagenesis induced by the compound to bescreened in the cell line to determine whether the compound hasmutagenic activity.

Furthermore, the invention also includes a method of screening acompound for the activity of inducing or inhibiting gene expressioncomprising the steps of:

(1) providing a hepatocyte cell line selected from the group consistingof Fa2N-4 and Ea1C-35;

(2) exposing the cell line to the compound to be screened; and

(3) determining the inhibititory or inductive effect of by the compoundto be screened in the cell line to determine whether the compound hasthe activity of inducing or inhibiting gene expression.

Similarly, the invention also includes a method of screening a compoundfor cytotoxicity comprising the steps of:

(1) providing a hepatocyte cell line selected from the group consistingof Fa2N-4 and Ea1C-35;

(2) exposing the cell line to the compound to be screened; and

(3) determining the extent of cytotoxicity induced by the compound to bescreened in the cell line by determining the viability of the cell lineas a function of either or both of concentration and time of exposure inorder to determine whether the compound has cytotoxic activity.

One of the most significant screening methods according to the presentinvention is a method of screening for drug-drug interactions. These canbe mediated by either a cytochrome P450 (CYP) enzyme or a multi-drugtransporter (MDR) protein, such as MDR1. These drug-drug interactions,where administration of one drug interferes with the metabolism of asecond drug, are of considerable clinical importance. They can occurwith prescription drugs or over-the-counter drugs.

One method for screening for the existence of a drug-drug interactionaccording to the present invention comprises the steps of:

(1) providing a hepatocyte cell line selected from the group consistingof Fa2N-4 and Ea1C-35;

(2) exposing the cell line to a first drug;

(3) determining whether the first drug induces the production of acytochrome P450 enzyme that can metabolize the second drug by measuringthe production of the cytochrome P450 enzyme induced by the first drugto screen for the existence of an interaction between the first drug andthe second drug.

Another method for screening for the existence of a drug-druginteraction comprises the steps of:

(1) providing a hepatocyte cell line selected from the group consistingof Fa2N-4 and Ea1C-35;

(2) exposing the cell line to a first drug;

(3) determining whether the first drug induces the production of a MDRprotein by measuring the production of the MDR protein induced by thefirst drug to screen for the existence of an interaction between thefirst drug and the second drug.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

EXAMPLES

The following examples are provided by way of describing specificembodiments of the present invention without intending to limit thescope of the invention in any way.

Example 1 Characterization of Immortalized Human Hepatocytes

Over 100 human hepatocyte clonal cell lines were established bytransfecting human hepatocytes with the simian virus 40 large T andsmall t antigen genes under control of the SV40 early promoter. Two celllines designated Ea1C-35 and Fa2N-4 are described.

Both cell lines were created by lipofection-mediated transfection ofprimary cryopreserved human hepatocytes with vectors containing the SV40largeT and small t antigens. The Ea1C-35 cell line was derived fromtransfection of cryopreserved human hepatocytes with the immortalizationvector pBlue Tag, a recombinant plasmid containing the early region ofwild-type SV40. The pBlue Tag vector was constructed as follows: pBR/SV(ATCC) was digested with restriction enzymes KpnI and BamHI to release a2995 bp fragment (239-2468 bp, numbering according to Fiers, W et alScience, 273:113-120) containing the SV40 early promoter and the codingregions from small t and large T antigens. This KpnI/BamHI fragment wasinserted into the pBluescript SK vector (Strategene) to producepBlueTag; a Bluescript based vector that uses the SV40 promoter to driveT antigen expression. Neomycin resistance was conferred on thetransfected cells as a selectable marker by co-transfection of a neoplasmid. Clones were initially selected based on their ability to growin G418 containing media. The Ea1C-35 cell line was established andmaintained in MCT's proprietary serum containing media, CSM.

The Fa2N-4 cell line was immortalized via lipofection-mediatedtransfection with a single immortalization vector. The early region ofthe SV40 genome, contained in the pBlueTag vector, was inserted into abackbone based upon the InvivoGen pGT60mcs plasmid and was named pTag-1.The T-antigen coding region is under the influence of a hybrid hEF1-HTLVpromoter. The vector also encodes a hygromycin resistance gene as a drugselectable marker. Clones were selected based on their ability to growin hygromycin containing media. The Fa2N-4 cell line was established andmaintained in MFE.

Example 2 Expression of Liver Specific Transcription Factors

Since retention of liver specific transcription factors is aprerequisite for expression of hepatic functions, clonal cell lines wereinitially screened by RT-PCR using primers for human HNF1, HNF3, HNF4α,HNF4,γ and C/EBP and albumin. Briefly, total RNA was prepared from 10⁶cells of each clonal cell line using the micro-isolation method ofBrenner et al. (55). Where is the information for the previousreference? 50 μg of E. coli rRNA (Sigma) was used as a carrier tofacilitate the isolation of RNA from a small number of cells. RT-PCRreactions were carried out using the Perkin Elmer Cetus, GeneAmp RNA PCRKit. One μg of total RNA was reverse transcribed using random hexamersand M-MLV reverse transcriptase according to the supplier'sinstructions. The PCR reaction was carried out using oligonucleotideprimers that defined nucleotide fragments unique for each transcriptionfactor. The primers were commercially synthesized and purified byCruachem (Fisher Scientific). The PCR reaction was carried out for 30cycles using an annealing temperature of 58° C. for 1 min. The PCRproducts were visualized in a 1% agarose gel after staining withethidium bromide. Positive control samples included RT-PCR analysis oftotal RNA of freshly isolated human hepatocytes (not shown). Both celllines expressed all five hepatocyte associated transcription factors, asshown below in Table 1. Albumin production was measured as an indicatorof hepatocyte specific gene expression. As shown below in Table 1, bothcell lines secrete albumin into the serum free conditioned medium asdetected by ELISA assay using an antibody that recognizes human albumin.TABLE 1 Albumin HNF- HNF- HNF- (μg/mg Clones HNF-1 3α 4α 4γ hC/EBPprotein) Fa2N-4 + + + + + 2.79 Ea1C-35 + + + + + 0.3

Example 3 SV40 Mediated Proliferative Activity

Primary human hepatocytes have limited proliferative activity whencultured. In order to overcome this characteristic, SV40 large T andsmall t antigens were introduced into the genome. The resulting clonalcell lines, Fa2N-4 and Ea1C-35 have subsequently been maintained inculture for up to 18 months. Both immortalized lines grow and functionwhen maintained in MFE medium and can be cryopreserved and banked.Indirect immunofluorescent staining using polyvalent antibodies againstlarge T antigen and albumin demonstrated that the cell lines continue toexpress the nuclear localized immortalizing gene FIG. 1 a) as well asexpress a hepatocyte specific gene characteristic of differentiatedfunction (FIG. 1 b). The morphology of the Ea1C-35 cell line is shownbelow (FIG. 1 c).

Example 4 Drug Metabolism Data

Both cell lines continue to catalyze Phase I (cytochrome P450) and PhaseII conjugative reactions in monolayer cultures based on the metabolismof model substrates. One of the most important Phase I enzymes isCYP3A4, which is responsible for the metabolism of approximately 50% ofall drugs. The expression of CYP3A4 can be modulated by many factorsincluding multiple drug intake that may induce or inhibit the overallexpression of this P450. Therefore the effective therapeutic dose of adrug is determined in part by CYP3A4 expression.

CYP3A4 modulators can be identified by monitoring the transcriptionalresponsiveness of the gene and by measuring enzymatic activity towardsmodel substrates (i.e. testosterone). For example, transcriptionalresponsiveness to prototypical pharmacological CYP3A4 inducers (i.e.rifampin) can be assayed by the reverse transcription polymerase chainreaction (RT-PCR) using specific primers to detect CYP3A4 mRNA.Rifampin-induced CYP3A4 enzymatic activity can also be measured by theproduction of the 6β-OH-testosterone metabolite when cells are incubatedwith testosterone. As shown below in Table 2, the Fa2N-4 cell line ismore sensitive to CYP inducers than the Ea1C-35 cell line.

In order to demonstrate that the cell lines continue to express Phase IIconjugating enzymes, cells were exposed to acetaminophen for 24 hoursand conditioned culture medium was collected and analyzed for theproduction of acetaminophen glucuronide or sulfate conjugates. Theproduction of the acetaminophen glucuronide and acetaminophen sulfateconjugates was measured by HPLC analysis. The results are shown in Table2. To determine the effect of passage number, the production ofacetaminophen glucuronide and acetaminophen sulfate was measured forFa2N-4 cells after 11, 14, 27, 32, 40, and 41 passages. For passage 41,ammonia clearance was also measured as an indicator of nitrogenmetabolism. The results are shown in Table 3. These results indicatethat both pathways are intact. TABLE 2 Characteristics of the Fa2N-4 andEa1C-35 cell lines Rifampin treated Control Rifampin CYP3A4 (μg 6β-OH(μg 6β-OH Acetaminophen Acetaminophen (mRNA fold testosterone/testosterone/ glucuronide sulfate Cell line induction)¹ mg protein)² mgprotein)² (μg/mg protein) (μg/mg protein) Fa2N-4 15.4 5.44 15.28 20.916.1 (p13) Ea1C-35 2.2 4.53 9.25 15 21.5 (p29)¹Cells were exposed to vehicle or rifampin for 72 hours. Data isexpressed relative to vehicle treated controls.²Cells were exposed to vehicle or rifampin for 72 hours and thenincubated with testosterone for 24 hours. Production of the6β-OH-testosterone metabolite was quantitated by HPLC analysis and datais expressed per mg total cell protein.

TABLE 3 Effect of Passage Number for Fa2N-4 Cells on Metabolism ofAcetaminophen Acetaminophen Acetaminophen Ammonia Glucuronide SulfateClearance (mg NH₃/ Cells Passage (μg/mg protein) (μg/mg protein) mgprotein/24 hr) Fa2N-4 11 15.18 ± 0.74 30.26 ± 0.31 14 16.43 ± 1.26 29.87± 1.83 27  7.93 ± 2.37 27.48 ± 2.2 32 10.42 ± 1.45 25.37 ± 0.84 40 12.68± 2.76 25.25 ± 1.99 41  21.4 ± 4.5  36.6 ± 1.2 246 ± 5.87

Example 5 Use of Immortalized Hepatocytes to Identify and Rank CYPInducers

Two lines of evidence indicate that immortalized human hepatocytes canbe employed to identify and rank CYP3A4 inducers based on ‘inductionpotency”. First, exposing Fa2N-4 cells to rifampin (10 μM) results in agreater production of the 6-β-OH-testosterone metabolite than treatingcells with weaker CYP3A4 inducers such as dexamethasone (50 μM) orphenobarbital (1 mM), as shown below in Table 4. Secondly, immunoblotanalysis demonstrated that exposure of each of the cell lines torifampin or phenobarbital for 48-72 hours increased expression of CYP3A4protein in comparison to vehicle-treated controls; however, exposure torifampin resulted in a greater increase expression of CYP3A4 protein.This data is shown in FIG. 2.

Example 6 Expression of Plasma Proteins by Fa2N-4 and Ea1C-35 Cell Lines

The utilization of immortalized human hepatocytes as proteinbiofactories requires that the cell lines continue to proliferate andsecrete plasma proteins when maintained in mass culture. In order toaddress this question, the expression of plasma proteins by these celllines was analyzed. The well-differentiated nature of these cell linesis further supported by their continued secretion of adult hepatocytefunction specific plasma proteins (FIG. 3). Culture medium was harvestedfrom Fa2N-4 and Ea1C-35 cells seeded into either 60 mm plates or rollerbottles and analyzed by Western blot analysis. Medium was concentrated50× by ultrafiltration and 40 μg of total protein was loaded per laneexcept for albumin (10 μg total protein/lane). Blots were incubated witheither monoclonal or affinity purified polyclonal antibodies againstalbumin, α-1-antitrypsin, Factor VIII and Factor IX and visualized usingsecondary antibodies conjugated to horseradish peroxidase followed byincubation with DAB substrate. As shown below in FIG. 3, both cell linescontinue to express albumin, α-1-antitrypsin, and Factor IX. Theexpression of Factor VIII was variable and highly dependent on cell lineand culture conditions. There was heterogeneity in the processing ofFactor IX, an observation also seen in the human plasma-derived protein.

As a confirmation, Fa2N-4 cells were grown to confluence in T-150 flasksin serum free medium and albumin production was measured by an ELISAassay. The results are shown in Table 4. The results indicate thatproduction of albumin persists through at least 41 passages of thecells. TABLE 4 Production of Albumin by Fa2N-4 Cells at DifferentPassage Numbers Albumin in Media Cells Passage (μg/ml) Fa2N-4 13 3.73 ±0.64 16 3.06 ± 0.11 33  9.5 ± 0.5 41 6.17 ± 0.29

Inter-α-inhibitor proteins (IαIp), natural serine protease inhibitorsfound in relatively high concentration in plasma have been shown to playroles in inflammation, wound healing and cancer metastasis reviewed byBost et al.[19]. IαIp is a family of plasma proteins made and secretedby hepatocytes. The major forms of IαIp are inter-α-inhibitor (IαI,containing one light chain peptide called bikunin and two heavy chains)and pre-a-inhibitor (PαI, containing one light and one heavy chain).Recently, a monoclonal antibody that recognizes the light chain of humanIαIp (MAb 69.31) was developed by scientists at Prothera Biologics.Using MAb 69.31 in a competitive ELISA, these investigators demonstratedthat plasma IαIp levels were significantly decreased in severe septicpatients compared to healthy controls (Lim Y P, Bendelja K, Opal S M,Siryaporn E, Hixson D C, Palardy J E. Correlation Between Mortality andthe Levels of Inter-alpha Inhibitors in Plasma of Severely SepticPatients. Journal of Infectious Disease, 188:919-926, 2003).

Western blot analysis, using MAb 69.31 revealed that both the Fa2N-4 andEa1C-35 cell lines continue to synthesize immunoreactive IαIp (data notshown). Subsequently, the amount of IαIp secreted into the conditionmedium was quantitated using an ELISA assay (see example below).

Example 7 Quantitative ELISA and Trypsin Inhibition Assay

The total trypsin inhibitory activity of the conditioned media includesthe activity from the major serine protease inhibitors, α-1-antitrypsinand IαIp. IαIp can be functionally and quantitatively measured using 1)an in vitro assay that measures inhibition of proteases such as trypsinand 2) a competitive ELISA using MAb 69.31, a monoclonal antibody fromProthera Biologics (providence, R.I.) that specifically recognizes humanIαIp), respectively. The trypsin inhibition assay is used to examine thebiological activity of serine protease inhibitors by using thechromogenic trypsin substrate L-BAPA(N(alpha)-Benzoyl-L-arginine-4-nitroanilide hydrochloride, FlukaChemicals). The assay is based on the ability of serine proteaseinhibitors to inhibit the hydrolysis of L-BAPA. Inhibition can bemonitored by a decrease in the rate of Δ absorbance/minute at 410 nm.The specific activity was calculated based on the biological activityper ug protein. Conditioned media of both hepatocyte cell lines Fa2N4and Ea1C35 were collected and concentrated 50× by using Amicon Ultraultrafiltration device with 30 kD cut off (Millipore). The total trypsininhibitory activity of the conditioned media includes the activity fromboth major serine protease inhibitors, α-1-antitrypsin and IαIp whichwere present in the hepatocyte conditioned media as detected by westernblot (see above) and ELISA assay (see below). The amount of IαIp in themedia was also measured in the competitive ELISA. The ELISA wereperformed as follows: 96 well Immunolon-4 plates (Dynex, USA) werecoated with purified IαIp (300 ng) in 50 mM carbonate buffer pH 9.6 andincubated overnight at 4° C. A serial dilution of purified human plasmaderived IαIp in PBS containing 1% rat serum was used to establish astandard curve. For the quantitative analysis of IαIp levels in culturemedia, 50 μL of media or serially diluted IαIp were added to individualwells of a 96 well plate. After the addition of 50 μL of MAb 69.31 toeach well, plates were incubated for 1 hr at 37° C. and subsequentlywashed using an automated plate washer (Labsystem). The bound MAb 69.31was detected by adding HRP-conjugated goat anti-mouse IgG (humanabsorbed) (Biosource, Camarillo, Calif., USA) for 1 hr at 37° C. Afterwashing, 100 μL 1-Step ABTS (Pierce, Rockford, Ill., USA) was added tothe wells and the absorbance at 405 nm was measured on ELISA platereader (BioTek). Each sample was tested in triplicate. Unconditionedculture media was used as baseline control. The results are shown inTable 5: TABLE 5 Trypsin Inhibition IαIp conc. Cultured Protein Conc.after 50x Activity after UF after UF media ultrafiltration (UF) [mg/mL][TIU/mg] [μg/mL] Ea1C35 4.50 115.0 20.08 Fa2N4 9.02 45.10 4.03

Example 8 Enzyme Induction in Fa2N-4 and Ea1C-35 Cell Lines

Induction of cytochrome P450 (CYP) and related drug metabolizing enzymes(including transporters) is a well recognized cause of clinicallysignificant drug interactions, as well as a cause of pharmacokinetictolerance or auto-induction (the process whereby a drug induces its ownhepatic metabolism) (1,2). Recent evidence implicates enzyme inductionas an important determinant of certain types of drug-inducedhepatotoxicity (3). Guidelines for assessing enzyme induction in vitrohave been outlined by Tucker et al. (4) and Bjorsson et al. (5). Thesetwo “consensus reports” identify primary cultures of human hepatocytesas the method of choice—the gold standard—for assessing theenyzyme-inducing potential of new chemical entities (NCE's) and drugcandidates. This in vitro approach, based on a human-derived testsystem, is superior to an in vivo approach based on tests in laboratoryanimals because drugs are known to cause enzyme induction in aspecies-specific manner (1). In fact, the two prototypical inducers usedin the studies described later in this Example, namely omeprazole andrifampin, are efficacious inducers of human CYP1A2 and CYP3A4 and yetthey do not induce the corresponding enzymes in rats or mice. The basicprocedures for conducting enzyme induction studies in primary culturesof human hepatocytes and Fa2N-4 cells are shown in the flowchart of FIG.4.

Following their attachment to collagen, hepatocytes are cultured for twodays, in accordance with the recommendations of the consensus reports(4,5). During this so-called adaptation period, the hepatocytes restoretheir normal hepatocellular morphology and function. Prior to thisredifferentiation, the hepatocytes are refractory to the enzyme-inducingeffects of drugs. Hepatocytes are treated with test articles andnegative and positive controls (i.e., solvent control and prototypicalinducers of human CYP enzymes) once daily for three consecutive days.Enzyme induction is assessed 24 hours after the last treatment by avariety of techniques, including mRNA analysis, western immunoblottingand/or measurement of enzyme activity either in the hepatocytesthemselves or, preferably, in microsomes prepared from the hepatocytes.The latter approach permits an in vitro-ex vivo comparison betweenmicrosomes isolated from hepatocytes (in vitro) and microsomes isolateddirectly from human liver (ex vivo). Measurement of enzyme activity isthe end-point advocated in both consensus reports (4,5).

The procedure for assessing enzyme induction in Fa2N-4 cells isremarkably similar to that described for human hepatocytes, asillustrated in FIG. 4. The Fa2N-4 cells are propagated on a collagensubstratum in a proprietary medium developed by MCT. This medium, calledMFE Support Medium F (formerly known as Multi-Function Enhancing (MFE)medium) is available from XenoTech. The cells are detached bytrypsinization, isolated by centrifugation, and re-attached to collagenin the desired format (e.g., 6-, 12-, 24- or 96-well plates). After atwo-day adaptation period, the cells are treated once daily for threeconsecutive days with test article or the appropriate negative andpositive controls (i.e., solvent controls and prototypical inducers ofhuman CYP enzymes). Enzyme induction is assessed 24 hours after the lasttreatment.

Morphologically, Fa2N-4 cells closely resemble human hepatocytes, asshown in FIG. 5. This is significant because normal hepatocellularmorphology is intimately linked with normal hepatocellular function;both reflect the expression of highly differentiated properties ofhepatocytes (7-9).

The consensus reports (4,5) recommend that assessment of enzymeinduction utilize measurements of enzyme activity, rather thanmeasurments of mRNA or immunoreactive protein levels, although theselatter end-points often provide valuable information about the mechanismof induction. For example, they can also reveal induction by compoundsthat inhibit CYP activity so strongly that their inhibitory effect maskstheir inductive effect, as recently reported for ritonavir, which isboth a CYP3A4 inhibitor and inducer (13). This issue will be discussedlater in the section on Chemical specificity of enzyme induction inFa2N-4 cells.

When conducting enzyme induction studies in human hepatocytes, XenoTechmeasures enzyme activity in microsomes. CYP activity measured inmicrosomes prepared from the cultured hepatocytes (in vitro activity)can be compared with that measured in microsomes prepared directly fromhuman livers (ex vivo activity). Such comparisons provide compellingevidence that XenoTech's technique for culturing human hepatocytessupport CYP enzyme activities that are comparable to those present inhepatocytes in vivo.

In order to prepare microsomes for such analysis, XenoTech cultureshuman hepatocytes in large (60 mm) dishes, which generally restrictssuch studies to an analysis of drug candidates that are fairly welladvanced in the drug development process. Fa2N-4 cells provide thecapability of analyzing the enzyme-inducing properties of larger numbersof new chemical entities (NCEs), allowing an assessment ofenzyme-inducing potential in preclinical drug development or discovery.With this goal in mind, XenoTech has focused on measuring enzymeinduction in Fa2N-4 cells in a variety of higher throughput formats,including 6-, 12-, 24- and 96-well plates. When cells are cultured undersuch conditions, it is impractical to prepare microsomes, hence, anassessment of induction based on measurements of enzyme activity mustinvolve adding marker substrates to the Fa2N-4 cells.

XenoTech assesses enzyme induction in Fa2N-4 cells by incubating thecells with phenacetin (to measure CYP1A2), bupropion (to measureCYP2B6), diclofenac (to measure CYP2C9) or midazolam (to measureCYP3A4). In each case, the final concentration of substrate is 100 μM.Metabolite formation is determined by assaying aliquots of the cellculture medium at various times (up to 8 hours) by LC/MS/MS. Tofacilitate a comparison of different CYP activities under a variety ofconditions, the results are expressed relative to control activitydetermined at the 8-hour time point.

Fa2N-4 cells respond appropriately to enzyme inducers. As in the case ofhuman hepatocytes, CYP1A2 is highly inducible by those agents thatactivate the Ah receptor, whereas those agents that activate PXR and/orCAR cause induction of CYP3A4 and, to a lesser extent, CYP2B6 andCYP2C9. As shown in FIG. 6, treatment of Fa2N-4 cells with 100 μMomeprazole causes marked induction of CYP1A2 activity, whereas treatmentwith 20 μM rifampin induces CYP3A4 and, to a lesser extent, CYP2B6 andCYP2C9 activity. For the experiment depicted in FIG. 6, the Fa2N-4 cellswere cultured in 6-well plates.

Enzyme induction in Fa2N-4 cells is reproducible from one experiment tothe next, and across different sized multi-well plates. FIG. 7 depictsthe results of a comparison of the reproducibility of induction ofCYP2B6 (bupropion hydroxylase) activity by rifampin, across threedifferent plate formats. In addition, reproducibility of CYP1A2 andCYP3A4 induction across multiple cell passages was assessed, and thoseresults are shown in FIG. 8. The reproducibility in magnitude ofinduction across passages 32-47 is excellent for both CYP enzymes, andis superior to the reproducibility of induction typically seen withindividual preparations of human hepatocytes. Preparations of humanhepatocyte from different individuals can demonstrate enormousvariability in magnitude of induction, under identical conditions. Thus,reproducibility of induction of multiple passages of the Fa2N-4 cells ismarkedly superior to reproducibility with the same, or different, freshhuman hepatocyte preparations.

It is not widely known, but enzyme induction in human hepatocytes isaffected by the format of the cell culture system, such that themagnitude of induction tends to decline and become less reproducible aswell size decreases. This feature (together with the limited and erraticsupply of human liver) complicates the use of human hepatocytes forhigher throughput screening in a 96-well format. This complication doesnot occur with Fa2N-4 cells.

Enzyme induction in Fa2N-4 cells was assessed in 6-, 12-, 24-and 96-wellplates. Induction of CYP2B6 by rifampin is the same in 6-, 12- and24-well plates, as shown in FIG. 9 (studies in 96-well plates are inprogress). Identical results were obtained with CYP2C9 (results notshown). FIG. 10 shows the effect of cell culture format on the inductionof CYP1A2 by omeprazole and the induction of CYP3A4 by rifampin. Cellculture format appears to influence CYP1A2 induction, in that themagnitude of induction was greater in a 6-well than in a 12-, 24- or96-well format. However, in all cases, omeprazole induced CYP1A2activity at least 9 fold over control. (Note: CYP1A2 induction in the96-well plate was probably greater than 9.3 fold because, in this case,phenacetin O-dealkylation was measured after one hour, which is notoptimal for measuring CYP1A2 activity, as shown in FIG. 6.) In the caseof CYP3A4, induction by rifampin is similar in the 6-, 12-, 24- and96-well format (FIG. 10).

Overall, the results in FIGS. 9 and 10 indicate that enzyme induction inFa2N-4 cells can be assessed in a variety of cell culture formats,including 96-well plates, which bodes well for higher throughputscreening of enzyme inducers.

The induction of CYP enzyme activity in Fa2N-4 follows an appropriateand anticipated time course. To achieve maximum induction of CYPenzymes, human hepatocytes are treated for three to five consecutivedays with test articles and prototypical inducers (positive controls),as recommended in the consensus reports (4,5). The time course of CYP1A2and CYP3A4 induction in Fa2N-4 cells is shown in FIG. 11. The resultsare similar to those observed in primary cultures of human hepatocytes(12).

Enzyme induction in Fa2N-4 cells occurs over an appropriate range ofinducer concentrations. The concentration-response curves for CYP1A2induction by omeprazole and for CYP3A4 induction by rifampin in Fa2N-4are shown in FIG. 12. Similar results are observed in human hepatocytes(12,13). For studies with human hepatocytes, 100 μM omeprazole and 20 μMrifampin can be routinely used to achieve maximum induction of CYP1A2and CY3A4, respectively. These same concentrations are recommended forinduction studies in Fa2N-4 cells. In the case of rifampin,concentrations above 20 μM caused less than maximum induction in Fa2N-4cells. A similar phenomenon has been observed in some preparations ofhuman hepatocytes (13) but not others (12).

Fa2N-4 cells respond appropriately to those compounds that do and thatdo not induce CYP enzymes in human hepatocytes. For example, compoundsshown previously to activate PXR and induce CYP3A4 in human hepatocytes(13) induce CYP3A4 activity in Fa2N-4 cells, whereas Ah receptoragonists do not, as shown in FIG. 13. An exception is clotrimazole,which is both a CYP3A4 inducer of enzyme biosynthesis and inhibitor ofenzyme activity. In this case, induction of CYP3A4 was masked by theinhibitory effect of clotrimazole (which is consistent with clinicalobservation). The results obtained with clotrimazole in Fa2N-4 cells arereminiscent of those observed in human hepatocytes treated withritonavir, which is also a CYP3A4 inhibitor and inducer (13). Whencompounds function as both inhibitor and inducer, it is helpful toassess enzyme induction by measuring both enzyme activity and eithermRNA or immunoreactive protein levels. This lesson applies to humanhepatocytes as well as Fa2N-4 cells.

Investigators at Pfizer (Mills et al. [14]) and Hoffmann-La Roche(Morris et al. [15]) have examined enzyme induction in Fa2N-4 cellsbased largely on measurements of mRNA levels, which were determined bythe Invader© cleavase assay (14) or by TaqMan RT-PCR (15). In bothstudies, induction of mRNA encoding CYP1A2, CYP2C9, CYP3A4 andP-glycoprotein (MDR-1) was measured in Fa2N-4 cells treated with avariety of inducers (rifampin, phenobarbital, dexamethasone,clotrimazole, β-naphthoflavone and chrysin in the case of Mills et al.and rifampin, phenobarbital and omeprazole in the case of Morris et al.)in 6-and 24-well plates (14) or in 6- and 96-well plates (15). Mills etal. (14) reported that β-naphthoflavone (10 μM) induces CYP1A2 mRNA upto 6 fold; rifampin (20 μM) induces CYP3A4 and CYP2C9 mRNA by up to 15and 3 fold, respectively, and phenobarbital (1 mM) induces 3A4 andCYP2C9 mRNA by 12 and 2.5-fold, respectively. Slightly less inductionwas observed in 24-well plates compared with 6-well plates. For example,rifampin induced CYP3A4 mRNA 15 fold in 6-well plates versus 9 fold in24-well plates. In general, the results described in this reportresemble those reported by Mills et al. (14), although a comparison ofthe two studies suggests that, in the case of CYP3A4, induction at themRNA level is greater than induction at the level of enzyme activity,whereas the converse appears to be true in the case of CYP1A2. However,comparing the results of studies conducted in different laboratories isnot straightforward, especially when different end-points are measured.In the study by Morris et al. (15), induction of CYP2C9 and CYP3A4 wasmeasured at both the activity and mRNA levels in a 96-well format. Inthe case of CYP3A4, mRNA levels increased more than activity (e.g., 10μM rifampin increased mRNA levels 11 fold versus a 7.7-fold increase inCYP activity) whereas the opposite was observed in the case of CYP2C9(mRNA increased 1.4 fold versus a 2.6 fold increase in CYP2C9 activity).Morris et al. (15) also demonstrated that induction of CYP3A was due toinduction of CYP3A4, not CYP3A5 (a predominantly kidney form) or CYP3A7(a predominantly fetal form), which supports the contention that Fa2N-4cells behave like well differentiated adult hepatocytes. Like Mills etal. (14), Morris et al. (15) demonstrated that treatment of Fa2N-4 cellswith rifampin (or high concentrations of phenobarbital) causes a ˜2-foldincrease in the mRNA encoding the transporters P-glycoprotein (MDR-1)and MRP2. Overall, there is excellent agreement between the studiesconducted at XenoTech and those conducted by Mills et al. at Pfizer (14)and Morris et al. at Hoffmann-La Roche (15).

Table 6 summarizes the magnitude of induction of CYP1A2, CYP2B6, CYP2C9and CYP3A4 in Fa2N-4 cells and primary cultures of human hepatocytes.The latter data are from XenoTech's recent publication (Madan et al.,2003). In the case of CYP1A2, the magnitude of induction in Fa2N-4 cellswas greater than the average fold induction in human hepatocytes. In thecase of CYP2B6, CYP2C9 and CYP3A4, the magnitude of induction in Fa2N-4cells was comparable to the median fold induction in human hepatocytes,but less than the average fold induction. Median induction differsconsiderably from mean induction in human hepatocytes because the latteris markedly affected by the occasional samples with extremely highvalues of fold induction. This is illustrated in FIG. 14 for CYP3A4induction, which ranges from zero (less than 1.5 fold) to 145 fold.TABLE 6 Comparison of CYP Enzyme Induction in Fa2N-4 Cells and HumanHepatocytes Fa2N-4 Average Human hepatocytes* Enzyme induction Averageinduction Human hepatocytes* (Inducer) (range) (range) Median inductionCYP1A2 20 fold 13 fold 8.4 fold (Omeprazole (9.3-29) (2-56) or BNF)**CYP2B6 2.5 fold 4.1 or 13 fold*** 2.9 or 8.5 fold (Rifampin) (2.0-3.9)(up to 14 or 71) CYP2C9 2.0 fold 3.5 fold 3.1 fold (Rifampin) (1.6-2.8)(1.5-10) CYP3A4 5.1 fold 10 fold 3.8 fold (Rifampin) (4.0-6.9) (0-145)*Data from Maden et al., Effects of prototypical microsomal enzymeinducers on cytochrome P450 expression in cultured human hepatocytes,Drug Metab. Dispos. 31: 421-431, 2003.**BNF (β-naphthoflavone) was the inducer for human hepatocytes, whereasomeprazole was the inducer for Fa2N-4 cells.***CYP2B6 activity based on 7-ethoxy-4-trifluoromethylcoumarinO-dealkylation (4 fold) or S-mephenytoin N-demethylation (13 fold).

FIG. 15 also shows the effect of enzyme inducers on CYP1A2 and CYP3A4activity in Fa2N-4 cells. The left panel shows the fold induction forCYP1A2, which catalyzes phenacetin O-dealkylation. The compounds testedwere omiprazole, 3-methylcholanthrene, lanoprazole, 1,2-benzanthracene,β-naphthoflavone, resveratrol, probenecid, benzo[a]pyrene, oltipraz,phenytoin, and benzo[e]pyrene. The right panel shows the fold inductionfor CYP3A4, which catalyzes midazolam 1′-hydroxylation. The compoundstested were rifampin, dexamethasone, hyperforin, phenobarbital,sulfinpyrazone, ciglitazone, phenytoin, efavirenz, troleandomycin,simvastatin, vitamin D3, probenecid, troglitazone, carbemazepine,tamoxifen, omeprazole, fexofenadine, 3-methylcholanthrene, andclotrimazole.

Although the mean fold induction of CYP3A4 in human hepatocytes is 10fold, the median induction, which is a more meaningful comparator, isabout 4 fold.

Fa2N-4 cells in culture are morphologically and functionally similar toprimary cultures of human hepatocytes. The response of this cell line toenzyme inducers closely resembles that observed in human hepatocytes,which are considered the in vitro system of choice—the gold standard—forassessing the enzyme-inducing potential of drug candidates. Fa2N-4 cellsoffer a number of advantages over human hepatocytes; some of which makeFa2N-4 cells a promising in vitro test system for higher throughputscreening of new chemical entities. In contrast to human liver, thesupply of which is limited and erratic, Fa2N-4 cells are available inunlimited supply. Induction of CYP enzyme activity in Fa2N-4 cells ismore reproducible than that in human hepatocytes. Furthermore, CYPinduction in Fa2N-4 cells can be measured in a variety of cell cultureformats, including 96-well plates, whereas this is not always possiblewith human hepatocytes. Primary cultures of human hepatocytes arecurrently acknowledged by regulatory agencies as being an appropriate invitro test system for assessing the enzyme-inducing potential of drugcandidates, provided the studies are conducted in accordance withrecommendations outlined in the consensus reports (4,5). The Fa2N-4 cellline is a new cell line with unique properties. As such, it is notapproved by regulatory agencies, but the similarity between Fa2N-4 cellsand primary cultures of human hepatocytes suggests that such approval isa future possibility.

This report focuses on enzyme induction in the Fa2N-4 cell line. Studiesat Pfizer by Mills et al. (14) suggest that the Ea1C-35 cell line canalso be used for enzyme induction studies, although the Ea1C-35 havehigher basal CYP enzyme activity, which may blunt the magnitude ofinduction. The recent study by Morris et al. at Hoffmann-La Rochesupports this observation (15). Both cell lines can be used to examinecompounds for their ability to cause cellular toxicity. In fact, it isdesirable to include one or two tests of cellular toxicity (e.g., enzymeleakage to assess membrane integrity and Alamar blue reduction to assessmitochondrial respiration) so that a true lack of enzyme induction canbe distinguished from a failure of enzyme induction to occur due tocellular toxicity.

Drug-induced liver toxicity is an important clinical problem, andseveral drugs have been withdrawn from the market because of theirability to cause rare but severe (even lethal) cases of hepatotoxicity.XenoTech scientists have completed a preliminary analysis of theresponse of the Fa2N-4 cells to different concentrations of knowntoxicants and non-toxicants, using the endpoints of compromised membraneintegrity, determined by release of intercellular proteins (αGST orLDH), and perturbation of mitochondrial respiration.

Results on the use of the immortalized hepatocytes in toxicity studiesare shown in FIG. 16. Treatment of cells with toxic concentrations (upto 100 μM) of several agents, namely 3-methylcholanthrene, methotrexate,menadione, rotenone, and troglitazone, caused a loss of membraneintegrity, resulting in the release into the medium of an intracellularenzyme, namely α-glutathione S-transferase (α-GST), which was measuredwith Biotrin High Sensitivity Alpha GST EIA (Biotrin International,Dublin, Ireland). In contrast, little or no α-GST was released fromFa2N-4 cells treated with non-toxic concentrations of omeprazole,acetaminophen, probenecid, felbamate, or rifampin. It should be notedthat some of these agents, such as acetaminophen, can cause clinicallysignificant liver toxicity, but only at high doses (and hence at muchhigher concentrations than those used in the study depicted in FIG. 16.)The toxicity data is also summarized in Table 7. TABLE 7 Comparison ofthe Toxicity of 22 Compounds in Fa2N-4 Cells and Primary HumanHepatocytes Cellular Response Non-toxic Compound Toxic Compound SameRifampin 3-Methylcholanthrene Phenobarbital Methotrexate PhenytoinRotenone Carbamazepine Efavirenz Troleandomycin Lansoprazole OmeprazoleProbenicid Felbamate Acetaminophen Ciglitazone SulfinpyrazoneSimvastatin Fexofenadine Different Troglitazone* Benzo[a]pyrene*Hyperforin* Menadione***Fa2N-4 cells more sensitive than human hepatocytes**Fa2N-4 cells less sensitive than human hepatocytes

The Fa2N-4 cells may offer a superior alternative to other systems foridentifying compounds with a high potential to cause clinicallysignificant hepatotoxicity.

As of this writing, XenoTech has detected CYP1A2, 2B6, 2C9and 3A4activity in Fa2N-4 cells, and even greater activity is present inEa1C-35 cells. The cell lines have been shown by MCT to conjugateacetaminophen with glucuronic acid. These findings suggest that one orboth cell lines may be useful in assessing the metabolic stability ofdrug candidates. However, the basal metabolic rate of both lines issufficiently low that we are unable to currently recommend their use instudies of metabolic stability. We are researching different approacheswith the Fa2N-4 or Ea1C-35 cells, which may result in availability ofimmortalized hepatocytes capable of properly supporting metabolicstability studies.

References for Example 8

1. A. Parkinson. Biotransformation of Xenobiotics. Chapter 6 in:Casarett and Doull's Toxicology. The Basic Science of Poisons .Sixthedition (Ed: C. D. Klaassen). McGraw Hill. New York, pp. 133-224, 2001.

2. R. Levy, K. Thummel, W. Trager, P. Hansten and M. Eichelbaum.Metabolic Drug Interactions. Lippincott, William & Wilkins,Philadelphia, Pa. 2000.

3. J. Zhang, W. Huang, S. Chua, P. Wei and D. D. Moore. Modulation ofacetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR.Science 298, 422-424, 2002

4. G. T Tucker, J. B. Houston and S-M. Huang. Optimizing drugdevelopment: Strategies to assess drug metabolism/transporterinteraction potential—toward a consensus Pharmaceutic. Res. 18:1071-1080, 2001

5. T. D. Bjornsson, J. T. Callaghan, H. J. Einolf, V. Fischer, L. Gan,S. Grimm, J. Kao, S. P. King, G. Miwa, L. Ni, G. Kumar, J. McLeod, S. R.Obach, S. Roberts, A. Roe, A. Shah, F. Snikeris, J. T. Sullivan, D.Tweedie, J. M. Vega, J. Walsh, S. A. Wrighton. The conduct of in vitroand in vivo drug-drug interaction studies: A PhRMA perspective. J. Clin.Pharmacol. 43: 443-469, 2003

6. A. Madan, R. A. Graham, K. M. Carroll, D. R. Mudra, L. A. Burton, L.A. Krueger, A. D. Downey, M. Czerwinski, J. Forster, M. D. Ribadeneira,L-S. Gan, E. L. LeCluyse, K. Zech, P. Robertson, P. Koch, L. Antonian,G. Wagner, L. Yu and A. Parkinson. Effects of prototypical microsomalenzyme inducers on cytochrome P450 expression in cultured humanhepatocytes. Drug Metab. Dispos. 31: 421-431, 2003.

7. E. LeCluyse, P. Bullock, A. Parkinson and J. Hochman. Cultured rathepatocytes. In: Models for assessing Drug Absorption and Metabolism.(Eds: R T Borchard, P L Smith and G Wilson). Chapter 9, pp. 121-160.Plenum Press, New York, 1996.

8. E. LeCluyse, P. Bullock and A. Parkinson. Strategies for restorationand maintenance of normal hepatic structure and function in long-termcultures of rat hepatocytes. Adv. Drug Delivery Rev. 22,133-186, 1996.

9. D. Mudra and A. Parkinson. Preparation of hepatocytes for drugmetabolism studies. In: Current Protocols in Toxicology. Unit 5.8. (Ed:M Maines, C Brad-field, L Costa, E Hodgson, D Reed and I G Sipes). JohnWiley & Sons, Inc. 2001

10. A. Madan, R. DeHaan, D. Mudra, K. Carroll, E. LeCluyse and A.Parkinson. Effect of cryopreservation on cytochrome P450 enzymeinduction in cultured rat hepatocytes. Drug Metab. Dispos. 27: 327-335,1999

11. E. LeCluyse, P. Bullock, A. Madan, K. Carroll and A. Parkinson.Influence of extracellular matrix overlay and medium formulation on theinduction of cytochrome P450 2B in primary cultures of rat hepatocytes.Drug Metab. Dispos. 27: 909-915, 1999

12. E. LeCluyse, A. Madan, G. Hamilton, K. Carroll, R. DeHaan and A.Parkinson. Expression and regulation of cytochrome P450 enzymes inprimary cultures of human hepatocytes. J. Biochem. Mol. Toxicol. 14:177-188, 2000

13. G. Luo, M. Cunningham, S. Kim, T. Burn, J. Lin, M. Sinz, G.Hamilton, C. Rizzo, S. Jolley, D. Gilbert, A. Downey, D. Mudra, R.Graham, K. Carroll, J. Xie, A. Madan, A. Parkinson, D. Christ, B.Selling, E. LeCluyse and L-S. Gan. CYP3A4 induction by drugs:Correlation between a pregnane X receptor reporter gene assay and CYP3A4expression in human hepatocytes. Drug Metab. Dispos. 30: 795-804, 2002

14. J. B. Mills, R. Faris, J. Liu, S. Cascio and S. M. de Morais S M. AnHTS assay for induction of enzymes and transporters using a humanhepatocyte clonal line and RNA detection. Drug. Metab. Rev. 34: 124,2002. Abstract 248. (Presented at the annual ISSX meeting, Orlando,Fla., October 2002).

15. A. L. Morris, E. Awwal and K. B. Frank. In vitro induction ofcytochrome P450s and drug transporters using the Fa2N-4 immortalizedhuman hepatocyte line. Drug. Metab. Rev. 35: 125, 2003. Abstract 249.(Presented at the annual ISSX meeting, Providence, R.I., October 2003).

Example 9 Induction of Drug Metabolism Enzymes and MDR1 Using the CellLine Fa2N-4

Drug metabolizing enzymes, including cytochrome P450s (CYPs), andtransporters are involved in the clearance of drugs. The CYPs carry outvarious drug metabolism reactions, including oxidations andhydroxylations. Drug-drug interactions involving drug metabolizingenzymes and transporters are of increasing interest due to reports ofadverse reactions and loss of efficacy (Baciewicz et al., 1987; Spina etal., 1996). During drug development, in vitro assays can be used toavoid inducers, and characterize drug-drug interaction potential due toincreased drug clearance by the liver. CYPs are involved in themetabolism of drugs, primarily in the liver. Induction of CYP3A geneexpression is caused by a variety of marketed drugs including rifampin,phenobarbital, clotrimazole, and dexamethasone (Meunier et al., 2000;Sahi et. al., 2000, Luo et al., 2002; Madan et al., 2003) and representsthe basis for a number of common drug-drug interactions. CYP1A2 isinducible by 3-methylcholanthrene, beta-naphthoflavone, andtetrachlorodibenzodioxin (Li et al., 1998; Breinholt et al., 1999;Meunier et al., 2000; Madan et al., 2003). CYP2C9 can be induced byrifampin and phenobarbital, however, the magnitude of induction is lessthan that for CYP3A4 (Li et al., 1997; Madan et al., 2003). Inducers ofthe UGT1A family include rifampin, chrysin, and betanaphthoflavone (Liet al., 1997, Abid et al., 1997; Breinholt et al., 1999). The MDR1 geneproduct P-glycoprotein (P-gp) is an important drug efflux transporter.Inducers of P-gp include rifampin, phenobarbital, clotrimazole, anddexamethasone (Schuetz et al., 1996; Geick et al., 2001; Sahi et. al.,2003).

The pregnane X receptor (PXR) is the major determinant of CYP3A generegulation by drugs and other xenobiotics (Lehmann et al., 1998;Bertilsson et al., 1998, Pascussi et al., 2003). In addition, PXRmediates induction of CYPs 2B6, 2C8/9, and 3A7, as well as the drugtransporters MDR1, OATP-C, BSEP and MRP2 (Pascussi et al., 2003, Tironaet al., 2003). Other nuclear hormone receptors involved in induction ofADMET endpoints include glucocorticoid receptor (GR) (CYP2B6, CYP2C8/9,CYP3A4/5), constitutive androstane receptor (CAR) (UGT1A, CYP2B6,CYP3A4, and CYP2C9) and peroxisome proliferator-activated receptor(PPAR) (CYP4A) (Ferguson et al., 2002; Pascussi et al., 2003). Acytosolic receptor, the aryl hydrocarbon (Ah) receptor, is involved inthe induction of the CYP1A subfamily (Whitlock et al., 1996).

The ability to evaluate CYP induction in human hepatocytes is highlydesirable because several drugs are known to induce CYP enzymes inhumans but not rats, and vice versa (Bertilsson et al., 1998; Moore andKliewer, 2000). For example, pregnenolone 16-alpha-carbonitrile inducesCYP3A in rats but not humans, whereas rifampin is a known inducer ofCYP3A in humans but not rats. Primary cultures of human hepatocytes havethe distinct advantage of exhibiting species-specific induction of CYPisoforms, but are dependent on the availability of fresh cells anddonor-to-donor variability. Cell lines such as HepG2, LS180, and LS174T,have been useful in studying induction of a limited subset of CYPs anddrug transporters (Schuetz et al., 1996; Li et al., 1998; Geick et al.,2001), but lack adequate response for other inducible targets (Silva andNicoll-Griffith, 2002).

Induction of drug metabolizing enzymes and drug transporters can bedetected at the mRNA level (Schuetz et al., 1996; Abid et al., 1997; Liet al., 1998; Ferguson et al., 2002). The Invade® assay (Kwiatkowski etal., 1999; Eis et al, 2001) quantifies transcript expression from totalRNA extracted from cultured cells. It is an isothermal detection of RNAand does not require a PCR amplification step. An overlap betweenoligonucleotides consisting of an upstream invasive deoxyoligonucleotideand a downstream deoxynucleotide probe are both annealed to the RNAtarget, followed by cleavage by a 5′ nuclease of the downstream probes.A second cleavage reaction utilizes a fluorescence resonance energytransfer (FRET) oligonucleotide that further amplifies the signal. Thisassay can differentiate between closely related RNA transcripts, such asin CYP subfamilies (Eis et al, 2001). For the current studies, we haveused the immortalized human hepatocyte cell line Fa2N-4. We havecharacterized these cells by studying their drug metabolizing enzymes,both at the level of the transcript and enzyme activity. We have alsostudied the induction potential of the Fa2N-4 cells by treating themwith a few prototypical inducers of the major drug metabolizing enzymesand monitoring changes in mRNA and enzyme activities. This Exampledescribes the utilization of the immortalized human hepatocytes Fa2N-4in combination with the mRNA detection Invader assay as a potentialmethod to predict clinical drug-drug interactions due to increase in thetranscription of genes encoding drug metabolizing enzymes ortransporters.

Methods

Chemicals

Phenobarbital(5-ethyl-5-phenyl-2,4,6-trioxohexahydropyrimidine),dexamethasone(9-alpha-fluoro-16-alpha-methylprednisolone),β-naphthoflavone(5,6-benzoflavone),rifampin(3-[4-methylpiperazinyliminomethyl]rifamycin SV),clotrimazole(1-[o-chloro-α-,α-diphenylbenzyl]-imidazole), and1-cyclohexyl-3-(morpholinoethyl)carbodiimide metho-p-toluenesulfonate,testosterone, 6-hydroxytestosterone, 7-ethoxyresorufin, resorufin,hydrocortisone, and diclofenac were purchased from Sigma (St. Louis,Mo.). 4′-hydroxydiclofenac was purchased from Gentest (Bedford, Mass.).[¹³C₆]-4′-OH-diclofenac was produced internally at Pfizer.

Induction of Fa2N-4 cells

This cell line originated from human hepatocytes isolated from a 12-yearold female donor and were immortalized via transfection with the Simianvirus 40 large T antigen as described above. Fa2N-4 cells (FIG. 17) wereobtained from MultiCell Technologies (Warwick, R.I.) and cultured asfollows. For RNA analysis, multiwell plates were pre-coated with a rigidcollagen complex composed of 2.75 mM1-cyclohexyl-3-(morpholinoethyl)carbodiimide metho-p-toluenesulfonateand 4% (v/v) Vitrogen 100 purified collagen (Cohesion, Palo Alto,Calif.) in sterile saline (0.9% NaCl). Excess collagen was removed priorto cell plating. For enzyme activity analysis, Biocoat type I collagenplates were used (Becton-Dickinson, Bedford, Mass.). Fa2N-4 cells wereplated at confluency in MFE media (MultiCell Technologies, Warwick,R.I.) supplemented with 100 units/ml penicillin, 100 μg/mL streptomycinand 10% fetal bovine serum (GIBCO BRL, Grand Island, N.Y.). Media wasreplaced with serum-free MFE media supplemented with 100 units/mLpenicillin and 100 μg/mL streptomycin after cell attachment(approximately 3 hours). Cells were kept in an incubator set at 37° C.,5% carbon dioxide, and 95% relative humidity. Media was replaced withfresh serum-free MFE media supplemented with 100 units/mL penicillin and100 μg/mL streptomycin every 24 hours. Treatment of cells with drug wasinitiated 48 hours after plating. For RNA quantification, cells wereexposed to drug for 48 hours. For enzyme activity studies, cells wereexposed to drug for 72 hours. Fa2N4 cells are depicted in FIG. 17. Phasecontrast image of confluent Fa2N-4 cells plated in 96-well Biocoat TypeI collagen plates (Becton Dickinson, Bedford, Mass.) in MFE media(MultiCell Technologies, Warwick, R.I.) at 200× magnification are shownin FIG. 17.

RNA Analysis

Total RNA was extracted from cells using the mini RNeasy kit accordingto instructions provided by the manufacturer (Qiagen, Valencia, Calif.).RNA (100 ng) was analyzed using the Invader® RNA assay reagent kitsaccording to instructions provided by the manufacturer (Third WaveTechnologies, Madison, Wis.). Statistical analysis for increased levelsof RNA in samples as compared to vehicle-treatment was conducted usingthe 2-sample, unpaired Student's t-Test, p<0.05 indicating significantdifferences. Statistical analysis for increased levels of RNA to comparemultiple treatments (>2 samples) was conducted using ANOVA analysis,p<0.05 indicating significant differences, for the purpose of rankordering multiple inducers.

Enzyme Activity

CYP3A4. Activity was determined by measuring the extent of6β-hydroxytestosterone formation from testosterone by mass spectrometry,essentially as described by Wood et al (1983) and Sonderfan et al (1987;1988), with the following modifications: Test drugs were washed fromcells by removing dosing media, replacing with fresh media, andincubating cells for 1 hour. After removing wash media, reactions werestarted with the addition of 250 μL MFE media containing 200 μMtestosterone to the tissue culture well. At 30 minutes, aliquots wereremoved for analysis via HPLC or LC/MS/MS. For LC/MS/MS analysis, thealiquot was mixed with 1 volume of acetonitrile spiked with 250 ng/mlhydrocortisone. Mass spectrometry was carried out with a Perkin Elmer200 HPLC system and a Micromass Quattro II detector. Samples wereinjected and were ionized utilizing the electrospray positive ion modein a mobile phase of 70:30 methanol: trifluoroacetic acid 0.02% (v/v) at0.20 ml/min (isocratic) and a Keystone Aquasil C18, 100·2.1 mm, 5 μmparticle size column. Some of the studies used an HPLC-UV assay fortestosterone metabolism, as follows: 200 μl of medium was mixed with 5μl of internal standard (IS) solution (20 μg/ml prednisolone inacetonitrile) and evaporated to approximately 50 μl. Samples (20 μl)were then injected on an Agilent 1100 HPLC system utilizing an AgilentZorbax Eclipse XDB-C8 column (4.6×150 mm) with UV detection at 254 nm.Mobile phase A consisted of 10 mM ammonium phosphate in water, andmobile phase B consisted of 100% acetonitrile. Initial conditions were35% B for 3 min, increasing to 65% B over 2 min, then at 10 min,returning to 35% B, for a total run time of 15 min. Retention times were2.8 min, 3.0 min, and 7.0 min for 6-β-hydroxy-testosterone,prednisolone, and testosterone, respectively. The standard curve for6-β-hydroxytestosterone was linear from 25 ng/ml to at least 1000 ng/ml.Peak area for 6-β-hydroxy-testosterone was normalized to IS, andreported as fold-change from DMSO-treated cells.

CYP2C9. Activity was determined by measuring the extent of4′-hydroxydiclofenac formation using the method of Leemann et al.(1993), modified as follows. Test drugs were washed from cells byremoving dosing media, replacing with fresh media, and incubating cellsfor 15 minutes. After removing wash media, reactions were started withthe addition of 250 μl MFE media containing 7.5 μM diclofenac to eachwell. Aliquots were removed at 60 minutes for LC/MS/MS analysis. Massspectrometry was carried out with a Perkin Elmer 200 HPLC system and aMicromass Quattro II detector. Samples were injected and were ionizedutilizing the electrospray positive ion mode in a mobile phase of 50:50acetonitrile: 0.1% formic acid in water (v/v) at 0.27 ml/min (isocratic)and a Phenomenex, Synergi Max RP, 50·2.0 mm, 4 μm particle column.

CYP1A2. Activity was determined by measuring the extent ofO-dealkylation of 7-ethoxyresorufin using the fluorometric method ofBurke et al. (1985), with minor modifications (Rodrigues and Prough,1991). Test drugs were washed from cells by removing dosing media,replacing with fresh media, and incubating cells for 15 minutes. Afterremoving wash media, reactions were started with the addition of 250 μLMFE media containing 7-ethoxyresorufin (20 μM) to each well. Aliquotswere removed at 15 minutes for fluorometric analysis. Metabolites werequantified by comparing measurements to standard curves. Theconcentration of protein for each cell treatment was determined withBiorad DC reagents (Hercules, Calif.) according to instructions providedby the manufacturer, using bovine serum albumin as standard. Values wereused to calculate enzyme activities as picomoles of metabolite permilligram protein per minute of incubation.

Results

Inductive Response of Fa2N-4 Cells to Known Inducers of DrugMetabolizing Enzymes and Drug Transporters

As illustrated in FIG. 18, Fa2N-4 cells are useful for monitoringseveral endpoints including CYP1A2, CYP2C9, CYP3A4, UGT1A, and MDR1using the known inducers rifampin (induces CYP2C9, CYP3A4, UGT1A, andMDR1), phenobarbital (induces CYP2C9, CYP3A4, and MDR1), dexamethasone(induces CYP3A4 and MDR1), and β-naphthoflavone (induces CYP1A2 andUGT1A). In FIG. 18, induction of CYP1A2, CYP2C9, CYP3A4, UGT1A, and MDR1transcripts in Fa2N-4 cells is shown. Fa2N-4 cells were plated in24-well plates and exposed to 0.1% DMSO vehicle (open bars), 10 μMrifampin (red bars), 1000 μM phenobarbital (blue bars), 50 μMdexamethasone (green bars), and 10 μM beta-naphthoflavone (black bars)for 48 hours is shown. The levels of transcripts were quantified fromtotal RNA isolated from the treated cells. Plot represents the mean±SDfrom the data of quadruplicate samples. Asterisk denotes statisticallysignificant increase in transcript versus vehicle control treatment(Student's t-Test, p<0.05). CYP3A4 inducers were significantly differentfrom each other using ANOVA analysis (<0.05). Increases in transcriptscan be observed for all positive controls. In comparison to the vehiclecontrol, CYP1A2 transcript was increased 15-fold after treatment with 10μM β-naphthoflavone, but not significantly increased with otherinducers. CYP2C9 transcript was increased 3.8-fold with 10 μM rifampin,2.6-fold with 1 mM phenobarbital, and not induced by treatment with 50μM dexamethasone, nor 10 μM beta-naphthoflavone. CYP3A4 transcript wasincreased 17-fold with 10 μM rifampin, 9.2-fold with 1 mM phenobarbital,and 1.3-fold with 50 μM dexamethasone. UGT1A transcript was increased2.1-fold with 10 μM beta-naphthoflavone, and not induced by treatmentwith 1 mM phenobarbital, nor 50 μM dexamethasone. Rifampin induction ofUGT1A was not statistically significant (p=0.08). MDR1 transcript wasincreased 3.1-fold with 10 μM rifampin, 2.3-fold induction with 1 mMphenobarbital, 1.3-fold induction with 50 μM dexamethasone, and therewas no MDR1 induction by 10 μM β-naphthoflavone. Table 8 summarizes theinduction data in Fa2N-4 cells for three CYPs expressed as fold-increasein mRNA compared to published data in primary hepatocytes. TABLE 8Summary of Reported Inductive Response in Fa2N-4 Cells as Compared toResponse of Primary Human Hepatocytes Fa2N-4 cells Primary cellsParameter Inducer Fold-increase Fold-increase CYP1A2 B-Naphthoflavone1.5 13 CYP2C9 Rifampin 3.8 3.5 Phenobarbital 2.6 1.8 CYP3A4 Rifampin 1710 Phenobarbitol 9.3 3.3CYP Enzyme Activity in Fa2N-4 Cells

CYP3A4 activity increased 8.9-fold and 2.1-fold, as assessed byincreases in formation of the 6-β-hydroxytestosterone with 10 μMrifampin and 50 μM dexamethasone, respectively, as compared tovehicle-treated control (FIG. 19A). In FIG. 19, measurement of inductionby cytochrome-450 enzyme activity is shown. Induction of CYP3A4, CYP2C9,and CYP1A2 enzyme activity in Fa2N-4 cells after 72 hour exposure to0.1% DMSO vehicle (VEH) or inducer is shown. Study was conducted using a12-well plate format. Data represents enzyme activity in terms ofmetabolite formed per milligram of total Fa2N-4 protein per minute ofincubation with parent compound. Data is from duplicate assays denotedby open and closed bars. (A) Measurement of CYP3A4 activity by formationof the testosterone metabolite 6-β-hydroxytestosterone in cells inducedwith vehicle, 10 μM rifampin (RIF), and 50 μM dexamethasone (DEX). (B)Measurement of CYP2C9 activity by formation of the diclofenac metabolite4′-hydroxydiclofenac in cells induced with vehicle, 10 μM rifampin(RIF), and 1000 μM phenobarbital (PB). (C) Measurement of CYP1A2activity by O-dealkylation of 7-ethoxyresorufin in cells induced withvehicle or 50 μM beta-naphthoflavone (BNF). Formation of4′-hydroxydiclofenac for assessment of CYP2C9 activity was increasedapproximately 2-fold for treatments with 10 μM rifampin and 1 mMphenobarbital (FIG. 19B). Fold changes in the EROD assay for CYP1A2 were27-fold with 10 μM β-naphthoflavone (FIG. 19C).

In addition to examining the inductive effect of a single concentrationof drug, the Fa2N-4 cells can also be used to look at dose-responserelationships. For example, EC50 values were calculated based on theresponse of Fa2N-4 cells dosed with multiple concentrations of rifampinranging from 100 nM to 50 μM. FIG. 20 contains EC50 plots for Fa2N-4cells using increased CYP3A4 transcript values FIG. 20A), as well asincreased CYP3A4 enzyme activity (FIG. 20B). In FIG. 20, dose-responsedependence of CYP3A4 induction by rifampin in Fa2N-4 cells is shown.Measurement of induction of CYP3A4 was performed in Fa2N-4 cells treatedwith 100 nM to 50 μM rifampin. Data was fitted using SigmaPlot (version8) using a 3-parameter sigmoidal curve. (A) Total RNA was analyzed todetermine level of CYP3A4 transcript and then compared to vehiclecontrol to determine fold-induction. Data represents mean±SD from thedata of triplicate samples. (B) CYP3A4 activity was measured byformation of the testosterone metabolite 6-β-hydroxytestosterone andthen compared to vehicle control to determine fold-induction. Datarepresents mean±SD from the data of triplicate samples. The calculatedEC50s were 0.43 μM (r²=92) and 0.77 μM (r²=94), for the transcript andenzyme activity, respectively. In addition, the calculated maximuminduction (Imax) values were 13-fold for the transcript endpoint and9.7-fold for the enzyme activity endpoint.

Fa2N-4 Inductive Response Over Multiple Passages

Multiple passages of the Fa2N-4 cells have been tested for CYP3A4induction. FIG. 21 shows response of multiple passages of Fa2N-4 cellsto a CYP3A4 inducer with a weak response (50 βM dexamethasone) and aCYP3A4 inducer that exhibits a strong response (10 μM rifampin). In FIG.21, various passages of Fa2N-4 cells were plated in 24-well plates andexposed to 0.1% DMSO vehicle (open bars), 50 μM dexamethasone (stripedbars), and 10 μM rifampin (black bars). (A) The levels of CYP3A4transcripts were quantified from isolated total RNA. Plot represents themean±SD from the data of quadruplicate samples. (B) CYP3A4 activity wasmeasured by formation of the testosterone metabolite6-β-hydroxytestosterone. Plot represents the mean of duplicate samples.All compounds showed statistically significant increase in transcriptversus vehicle control treatment (Student's t-Test, p<0.05). Treatmentwith dexamethasone increased CYP3A4 transcripts, 1.6-fold and 1.5-foldat passages 21 and 36, respectively. Treatment with 10 βM rifampinincreased CYP3A4 transcripts, 17-fold and 16-fold at passages 21 and 36,respectively (FIG. 21A). CYP3A4 enzyme activity was increased 2.1-foldand 2.0-fold for dexamethasone and 8.9-fold and 4.9-fold for 10 μMrifampin at passages 28 and 36, respectively (FIG. 21B).

Capacity for HTS with Fa2N-4 Cells and Invader® Assay

FIG. 22 compares various multiwell plate formats. In FIG. 22, inductionof CYP3A4 transcript in Fa2N-4 cells after 48 hour exposure to 10 μMrifampin (closed bars) is shown in comparison with vehicle (open bars).Data is from studies conducted in each multiwell plate format asindicated. Plot represents the mean±SD from the data of quadruplicatesamples. All compounds showed statistically significant increase intranscript versus vehicle control treatment (Student's t-Test, p<0.05).Regardless of the plate format, Fa2N-4 cells exhibit substantial CYP3A4inductive response to rifampin. Fold changes in CYP3A4 transcript were17.1-fold when using a 24-well plate, 6.6-fold when using a 24-wellplate, and 5.7-fold for when using a 96-well plate.

Discussion

The Fa2N-4 cells have the ability to induce CYP1A2, CYP2C9, CYP3A4,UGT1A, and MDR1 mRNA in response to known inducers. Using CYP3A4transcript as an endpoint, we have demonstrated the ability of the assayto rank inducers according to potency and demonstrate dose-response forrifampin as previously observed in primary human hepatocytes (Li et al.,1997; Sahi et al., 2000). In addition to distinguishing inducers fromnoninducers, this assay has a wide dynamic range for some endpoints suchas CYP3A4 and CYP1A2, enabling rank ordering for induction potency. Thesame decreasing potency for CYP3A4 inducers(rifampin>phenobarbital>dexamethasone) has been previously reported inthe literature for studies in primary human hepatocytes using both mRNAand enzyme activity endpoints (Luo et al., 2002). Our results using mRNAinduction in Fa2N-4 cells are in good agreement with the publication byMadan et al. (2003), who reported the effects of prototypical inducersfor CYP1A2, CYP2C9, and CYP3A4 in cultured primary human hepatocytes bymeasuring enzyme activity in microsome preparations from treated cells.Our induction results using the immortal cell line Fa2N-4 and mRNAmeasurements are in good agreement with the publication by Madan et al.(2003) using primary human hepatocytes. Madan et al. used livers fromseveral different donors and measured enzyme activity towards severalCYPs, after treatment with the same prototypical inducers as in ourstudy. The rank order for CYP3A4 induction potency for rifampin andphenobarbital expressed as fold induction over vehicle controls was thesame in both studies. CYP2C9 was also induced, albeit to a lesser extentthan CYP3A4, and CYP1A2 had the highest response in both systems.Another study (Sahi et al. 2000) using primary human hepatocytesreported EC50 for rifampin in CYP3A4 induction, using an activity assay.Those results are also in good agreement with our own EC50 results usingthe Fa2N-4 cells and mRNA measurements.

This immortal hepatocyte clone was identified in a screen of several ofclones where the best response to rifampin induction of CYP3A4 was theselection criteria. Hence, the most appropriate utilization of thisassay is for CYP3A4 induction. Further characterization of this cloneindicated that it had high response to a CYP1A2 inducer, enabling alsothe detection of this endpoint in a screening format. The dynamic rangeof the responses to CYP2C9 and MDR1 were smaller, but they were in thesame proportion as the inductive response found in fresh hepatocytes (Liet al., 1997; Madan et al. 2003; Sahi et al. 2003).

Although the average UGT1A transcript was higher in rifampin-treatedFa2N-4 cells than in vehicle-treated cells, the level of induction wasnot statistically significant. Previous induction studies in primaryhuman hepatocyte cite inter-individual variation in the effects ofrifampin, using 1-naphthol glucuronidation as an endpoint. Abid et al.(1997) reported that the variability may be attributed to differentialinduction of two UGT1A isoforms. The Invader® UGT1A oligonucleotidesused here span a common region in the RNA among all isoforms, andmeasurement of mRNA is a sum of all UGT1A isoforms. Thus, the inductiveeffect on rifampin could have been minimized by the non-induced UGT1Aisoforms. It is likely that probes designed for individual UGT1Aisoforms would be able to detect significant increases in their mRNA.

Induction in the Fa2N-4 cells is not limited to mRNA and can also beassessed at the enzyme activity level. The extent of induction usingmRNA quantified with Invader® correlated well with enzyme activity dataas indicated by similar rank order for several prototypical inducers.The ability to induce CYP enzyme activity provides further evidence onthe expression of a comprehensive array of CYPs in the Fa2N-4 cells. Inaddition, it shows the potential of these cells for alternativeapplications, such as CYP inhibition or metabolite generation.

The Invader® assay can adequately quantify induction based on mRNA levelincreases. Advantages of the mRNA endpoint include increased throughputand target specificity. For enzyme activity, a separate well in amultiwell plate must be used for each enzyme activity endpoint, whereasa single well can be used to assess multiple mRNA targets. The recoveryof RNA from each sample (24-well plate) is high enough to run up to 50separate mRNA endpoints. The Invader assay is able to discriminate amongclosely related CYPs, whereas enzyme activity assays are not alwaysspecific. For example, O-dealkylation of 7-ethoxyresorufin characterizesthe combination of CYP1A1 and CYP1A2, and 6-β-hydroxytestosterone canalso be formed by both CYP3A4 and CYP3A5 (Williams et al., 2002).

In contrast to fresh human hepatocytes, Fa2N-4 cells are readilyavailable. Since accessibility to fresh human hepatocytes is reliant onavailability of a suitable liver tissue donor, it can take a long timeto conduct experiments using hepatocytes isolated from three differentlivers to verify that a certain compound is an inducer. In addition,plating efficiency of fresh hepatocytes is unpredictable, so it is notuncommon to have a suitable donor, but find that the cells are notusable due to poor plating efficiency or substandard cell health. Fa2N-4cells can be passaged and used over several passages while retainingactivity of the major drug metabolizing enzymes. With fresh humanhepatocytes, cells can only be used one time, making it difficult tocompare data between studies. Plateable cryopreserved primary humanhepatocytes are an improvement by theoretically allowing multipleexperiments at different times from a single donor, or potentially theuse of multiple donors at one time. However, plateable cryopreservedprimary human hepatocytes are in limited supply. Both fresh primaryhuman hepatocytes and plateable cryopreserved primary human hepatocyteshave donor-to-donor variability, based on the influence of genetics, theenvironment, and co-medications. There are vast differences seen in thedrug metabolizing enzyme profile of donors, leading to the currentrecommendation of obtaining data from three donors before reaching aconclusion for induction potential of a chemical. In addition, someauthors cite the necessity for potency indexes in order to compare databetween donors (Silva and Nicoll-Griffith, 2002). The potency indexstandardizes data between donors by reporting the ratio of inductionresponse (i.e. fold-induction) of the test compound to that of aprototypical inducer.

Thus, our preliminary data using a few prototypical inducersdemonstrates that Fa2N-4 cells can be a suitable substitute for freshhuman hepatocytes in induction studies, and provide the additionalattribute of being amenable for higher throughput studies. Fa2N-4 cellsare superior to previously published immortal cell lines, as they showinduction of a varied number of genes. These cells can be used todetermine the induction potential of a drug, with findings consistentwith monitoring increased enzyme activity in primary human hepatocytes.Higher throughput cell culturing and analysis via mRNA endpoint enablesmore compounds to be tested and reduces the cost per compound; twofavorable traits for drug discovery assays. This induction assay has thepotential of becoming a useful tool for pharmaceutical companies toeliminate compounds with drug-drug interaction potential and tounderstand the likelihood and extent of DDI for compounds indevelopment.

References for Example 9

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Example 10 Reverse Transcription-Polymerase Chain Reaction Analysis forExpression of mRNA Transcripts After Exposure of Immortalized HepatocyteCell Lines to Inducers of Cytochrome P450

RT-PCR analysis was performed on the two immortalized human hepatocytecell lines designated Ea1C-35 and Fa2N-4. Cells were plated on type Icollagen coated dishes and maintained in MultiCell's proprietary media.Cultured cells were treated with rifampin (10 μM) for 3 days or an equalvolume of DMSO (e.g. Control).

This was done to screen for expression of hepatocyte specifictranscription factors (e.g. HNF-1α, HNF-3, HNF-4α, HNF4γ, cEBP), liverspecific genes (e.g. albumin and asialoglycoprotein receptor),transcription factors controlling drug metabolizing genes (e.g. SXR,RXRα, RXRβ, CAR) and phase I and phase II drug metabolizing enzymes(e.g. CYP1A2, CYP2A6, CYP2C9, CYP3A4, CYP2D6, CYP2E1, and UGT 1A1, UGT2B4, respectively).

Analysis was performed with and without exposure to rifampin, a knownpharmacological inducer of CYP3A4 expression. RT-PCR analysis revealedthat all transcripts examined were expressed by both cell lines but tovarious levels. Rifampin induction increased the expression of CYP3A4transcripts.

The following primers were used for the RT-PCR analysis: Albumin,Asialoglycoprotein II receptor, HNF-1α, HNF-3, HNF-4α, HNF4γ, c/EBP, UGT1A1, UGT 2B4, SXR, RXRα, RXRβ, CAR, CYP1A2, CYP2A6, CYP2C9, CYP3A4,CYP2D6, CYP2E1, Cytochrome c, and NADPH. The figure legends for gels 1-4(FIGS. 23-26) are given in Table 9. The figure legends for gels 5-8(FIGS. 27-30) are given in Table 10. TABLE 9 #1 PCR Product Gel #1 Gel#2 Gel #3 Gel #4 #2 Ea1C-35 p17, DMSO Ctrl UGT 1A1 SXR HNF-1α Albumin #3Ea1C-35 p17, Rifampin UGT 1A1 SXR HNF-1α Albumin #4 Fa2N-4 p34, DMSOCtrl UGT 1A1 SXR HNF-1α Albumin #5 Fa2N-4 p34, Rifampin UGT 1A1 SXRHNF-1α Albumin #6 #7 Ea1C-35 p17, DMSO Ctrl UGT 2B4 RXRα HNF-3 ASGPR II#8 Ea1C-35 p17, Rifampin UGT 2B4 RXRα HNF-3 ASGPR II #9 Fa2N-4 p34, DMSOCtrl UGT 2B4 RXRα HNF-3 ASGPR II #10 Fa2N-4 p34, Rifampin UGT 2B4 RXRαHNF-3 ASGPR II #11 #12 Ea1C-35 p17, DMSO Ctrl CAR RXRβ HNF-4α GAPDH #13Ea1C-35 p17, Rifampin CAR RXRβ HNF-4α GAPDH #14 Fa2N-4 p34, DMSO CtrlCAR RXRβ HNF-4α GAPDH #15 Fa2N-4 p34, Rifampin CAR RXRβ HNF-4α GAPDH #16#17 Ea1C-35 p17, DMSO Ctrl c/EBP GAPDH HNF-4γ #18 Ea1C-35 p17, Rifampinc/EBP GAPDH HNF-4γ #19 Fa2N-4 p34, DMSO Ctrl c/EBP GAPDH HNF-4γ #20Fa2N-4 p34, Rifampin c/EBP GAPDH HNF-4γ kit Ctrl

TABLE 10 PCR Product Gel #5 Gel #6 Gel #7 Gel #8 #1 100 bp marker 5 ul 5ul 5 ul 5 ul #2 Ea1C-35 p17, DMSO Ctrl CYP 3A4 CYP 2D6 GAPDH, RT(+), 60°C. Kit Ctrl, 61° C. #3 Ea1C-35 p17, Rifampin CYP 3A4 CYP 2D6 GAPDH,RT(+), 60° C. Kit Ctrl, 60° C. #4 Fa2N-4 p34, DMSO Ctrl CYP 3A4 CYP 2D6GAPDH, RT(+), 60° C. Kit Ctrl, 59° C. #5 Fa2N-4 p34, Rifampin CYP 3A4CYP 2D6 GAPDH, RT(+), 60° C. Kit Ctrl, 60° C. #6 #7 Ea1C-35 p17, DMSOCtrl CYP 2C9 CYP 2E1 GAPDH, RT(+), 61° C. #8 Ea1C-35 p17, Rifampin CYP2C9 CYP 2E1 GAPDH, RT(+), 61° C. #9 Fa2N-4 p34, DMSO Ctrl CYP 2C9 CYP2E1 GAPDH, RT(+), 61° C. #10 Fa2N-4 p34, Rifampin CYP 2C9 CYP 2E1 GAPDH,RT(+), 61° C. #11 #12 Ea1C-35 p17, DMSO Ctrl CYP 1A2 Cyto c GAPDH,RT(+), 59° C. #13 Ea1C-35 p17, Rifampin CYP 1A2 Cyto c GAPDH, RT(+), 59°C. #14 Fa2N-4 p34, DMSO Ctrl CYP 1A2 Cyto c GAPDH, RT(+), 59° C. #15Fa2N-4 p34, Rifampin CYP 1A2 Cyto c GAPDH, RT(+), 59° C. #16 #17 Ea1C-35p17, DMSO Ctrl CYP 2A6 NADPH GAPDH, RT(−), 60° C. #18 Ea1C-35 p17,Rifampin CYP 2A6 NADPH GAPDH, RT(−), 60° C. #19 Fa2N-4 p34, DMSO CtrlCYP 2A6 NADPH GAPDH, RT(−), 60° C. #20 Fa2N-4 p34, Rifampin CYP 2A6NADPH GAPDH, RT(−), 60° C.

Example 11 Conditions for the Expression of Plasma Proteins by Fa2N4 andEa1C35 Cell Lines

Two-dimensional gel electrophoretic analysis was used to separate thesecreted proteins of the Fa2N4 and Ea1C35 cell lines. Using Invitrogen'sZOOM IPGRunner system, the first IEF separation of the proteins wascarried out using fixed pH gradient strip (pH range of 3-10) followed bythe second dimension separation using 4-12% Tris-Gycine SDS-PAGE. Inboth cell lines multiple spots of proteins could be identified aspossible candidates for therapeutic proteins. (FIG. 31A. Fa2N4; 31B,Ea1C35). After the 2-dimensional gel separation the secreted proteins ofthe Ea1C35 cell line were transferred onto nitrocellulose and Westernblot analysis using anti-Factor IX antibody was performed. Reactiveprotein with MW of 70 kD and pI 6.5-7.0 was detected (FIG. 31C).

Immunostaining of Fa2N-4 cells for albumin expression was also carriedout. Cells were plated on type I collagen and cultured in serum freemedium for 72 hr. Albumin was visualized by indirect immunofluorescencewith a fluorescein conjugated secondary antibody. As shown in FIG. 1 b,virtually all of the cells express albumin (e.g. green color).

In order to determine if the Ea1C-35 and Fa2N-4 cells from variouspassages made and secreted transferrin, the cells were cultured in serumfree medium without transferring for 7 days. Conditioned culture mediumwas collected after 7 days and immunoblot analysis was performed using acommercially available antibody against transferrin. Human plasma wasused as the positive control. Immunoblots revealed that the cells fromall passages continue to express this plasma protein. The results areshown in FIG. 32. The lanes for FIG. 32 are shown in Table 11. TABLE 11Lane # Samples 1 Marker 2 Ea1C-35 p15 3 Ea1C-35 p24 4 Ea1C-35 p29 5Ea1C-35 p43 6 Fa2N-4 p10 7 Fa2N-4 p23 8 Fa2N-4 p31 9 Fa2N-4 p39 10 Humanplasma

The economical production of therapeutic plasma proteins using culturedimmortalized human hepatocytes as producer cells can only beaccomplished if the cells continue to make and secrete these plasmaproteins when expanded in mass culture. In order to initially evaluatethis question, Fa2N-4 cells were grown to confluence in T25, T75 andT150 culture flasks and selected plasma proteins were quantitated usingELISA assays in combination with capture antibodies that recognizedalbumin, AAT or IαIp. An equivalent number of cells were initiallyplated per square cm, 5, 15 and 30 million cells, respectively.Conditioned medium was collected for 3 days, pooled, concentrated 10× byultrafiltration and assayed. As shown in Tables 12-14, the totalexpression of each plasma protein was approximately proportional to cellnumber. Values represent the mean±SD for triplicate samples. Over the3-day period cells cultured in T150 flasks produced approximately 200 ugalbumin, 500 ng IαIp and 150 ng AAT.

We plan to use immortalized human hepatocytes as biofactories for thecommercial production of therapeutic proteins. Therefore, it isessential that plasma protein secretion must not be significantlydecreased in long-term culture. We recently initiated a study in orderto evaluate this question, Fa2N-4 cells were grown in T25, T75 and T150culture flasks as described above and albumin production was measured asan indicator of overall protein secretion. Conditioned medium wascollected on Day 3, cells were refed and resampled on Day 6. Albuminsecretion was analyzed by an ELISA assay. The results indicate thatalbumin secretion continues to increase over the 6 day collection periodirrespective of the plating format (see Table 15). Of particular note,there is a dramatic increase in albumin when cells were cultured in theT75 and T150 flasks. Since total cellular protein does not significantlyincrease with time in culture (data not shown), it seems likely thatthese results are due to enhanced production as a result of adaptationto culture conditions and not the result of a dramatic increase in cellnumber per flask.

Since the production of some plasma proteins can be modulated by acutephase proteins such as TNFα in vivo we reasoned that this cytokine mightenhance the secretion of plasma proteins by immortalized humanhepatocytes. In the present study, we examined the effects of TNFα onthe secretion of AAT. Fa2N-4 cells were maintained in serum freeproprietary MFE media containing TNFα (0, 1, 5, or 10 ng/ml) for 3 days.The results are shown in Table 16. Values are the average of duplicatesamples. As shown in Table 16, the secretion of AAT was most notablyincreased by the inclusion of 5 ng/ml TNFα in the serum-free culturemedium. Therefore, it might be possible to increase AAT production usingthis cytokine. TABLE 16 Concentration of Antitrypsin Antitrypsin SampleTumor Necrosis Factor Alpha (ng)/μg Protein (ng)/well #1 TNF 0 ng/ml0.21 14.00 #2 TNF 1 ng/ml 0.34 21.00 #3 TNF 5 ng/ml 0.43 44.73 #4 TNF 10ng/ml 0.48 32.93

Albumin expression is regulated in part by a dexamethasone induciblepromoter. In order to examine the effects of dexamethasone on theproduction and secretion of albumin by immortalized human hepatocytes,Fa2N-4 cells (passage 32) were cultured on type I collagen dishes withor without dexamethasone in the culture medium for 48 hrs and albuminexpression was measured by an ELISA assay. Values represent the averageof duplicate samples. As summarized in the table below (Table 17), thesecretion of albumin was significantly decreased in the absence ofdexamethasone. TABLE 17 Concentration of Albumin Dexamethasone (μg/ml) 040.0 1.0 μM 100.0

Example 12 Ability to Produce and Express Therapeutic Plasma Proteins

The ability of our Fa2N-4 cell line to correctly produce animmunologically reactive therapeutic plasma protein was illustrated withthe production of immuno-reactive human growth hormone (hGH). On the dayprior to transient transfection, Fa2N-4 cells were plated at a densityof 0.5-0.8×10⁶ cells per well in six-well Nunc plates using 10% NBCS-MFEmedium. On the day of transfection the cells were washed one time toremove serum and a CMV-based plasmid, containing the complete cDNA forhGH, was transiently transfected into the Fa2N-4 cells using either anInvitrogen Lipofectamine Plus or a Qiagen Effectene transfection reagentkit. The transfections were performed as per the manufacturers'protocols.

Conditioned media was withdrawn from each well after 24 and/or 48 hoursand was subsequently used for an ELISA-based immunodetection assay. TheELISA assay is a colorimetric enzyme immunoassay for the quantitativedetermination of secreted hGH utilizing the sandwich ELISA principle.Microtiter plate pre-bound antibodies to hGH bind to secreted hGHcontained in the conditioned media. Subsequently, a digoxigenin labeledhGH antibody binds to a second epitope of the hGH peptide contained inthe conditioned media and retained on the microtiter plate. An antibodyto digoxigenin, which is conjugated to peroxidase is then added andfollowed by the peroxidase substrate ABTS. The peroxidase-catalyzedcleavage of the substrate yields a colored reaction product that can beeasily detected using a microtiter plate reader.

Our results confirm that using either transfection kit and harvestingthe conditioned media at either 24 or 48 hours post-transfection, theFa2N-4 cells produce extraordinarily large quantities of doubleimmunodetected hGH while transfection with LacZ or no plasmid negativecontrols produced no detectable levels of hGH. A photograph of the ELISAplates 1 and 2 are shown in FIG. 33 and FIG. 34, respectfully. The keyfor FIGS. 33 and 34 is shown in Table 18. TABLE 18 1 2 3 4 5 6 7 8 9 1011 12 Plate 1 A Blank Std 0 Std 80 1QLacZ 2 1Q(1:10)3 1Q(1:30)1 1Neg2′1Q(1:10)3′ 1Q(1:30)1′ 2QLacZ 2 2Q(1:10)3 2Q(1:30)1 B Blank Std 0 Std 801QLacZ 2 1Q(1:10)3 1Q(1:30)1 1Neg2′ 1Q(1:10)3′ 1Q(1:30)1′ 2QLacZ 22Q(1:10)3 2Q(1:30)1 C Blank Std 10 Std 160 1QLacZ 3 1Q(1:20)1 1Q(1:30)21Neg3′ 1Q(1:20)1′ 1Q(1:30)2′ 2QLacZ 3 2Q(1:20)1 2Q(1:30)2 D Blank Std 10Std 160 1QLacZ 3 1Q(1:20)1 1Q(1:30)2 1Neg3′ 1Q(1:20)1′ 1Q(1:30)2′ 2QLacZ3 2Q(1:20)1 2Q(1:30)2 E Blank Std 20 Std 320 1Q(1:10)1 1Q(1:20)21Q(1:30)3 1Q(1:10)1′ 1Q(1:20)2′ 1Q(1:30)3′ 2Q(1:10)1 2Q(1:20)2 2Q(1:30)3F Blank Std 20 Std 320 1Q(1:10)1 1Q(1:20)2 1Q(1:30)3 1Q(1:10)1′1Q(1:20)2′ 1Q(1:30)3′ 2Q(1:10)1 2Q(1:20)2 2Q(1:30)3 G Blank Std 401QLacZ 1Q(1:10)2 1Q(1:20)3 1Neg1′ 1Q(1:10)2′ 1Q(1:20)3′ 2QLacZ 12Q(1:10)2 2Q(1:20)3 2Neg1′ 1 H Blank Std 40 1QLacZ 1Q(1:10)2 1Q(1:20)31Neg1′ 1Q(1:10)2′ 1Q(1:20)3′ 2QLacZ 1 2Q(1:10)2 2Q(1:20)3 2Neg1′ 1 Plate2 A Blank 2Neg3′ 2Q(1:20)1′ 2Q(1:30)2′ ILacZ 3 I(1.0)1 I(2.0)2 Blank′ BBlank 2Neg3′ 2Q(1:20)1′ 2Q(1:30)2′ ILacZ 3 I(1.0)1 I(2.0)2 Blank′ CBlank 2Q(1:10)1′ 2Q(1:20)2′ 2Q(1:30)3′ I(0.5)1 I(1.0)2 I(2.0)3 Blank′ DBlank 2Q(1:10)1′ 2Q(1:20)2′ 2Q(1:30)3′ I(0.5)1 I(1.0)2 I(2.0)3 Blank′ EBlank 2Q(1:10)2′ 2Q(1:20)3′ ILacZ 1 I(0.5)2 I(1.0)3 Blank′ F Blank2Q(1:10)2′ 2Q(1:20)3′ ILacZ 1 I(0.5)2 I(1.0)3 Blank′ G Blank 2Neg2′2Q(1:10)3′ 2Q(1:30)1′ ILacZ 2 I(0.5)3 I(2.0)1 Blank′ H Blank 2Neg2′2Q(1:10)3′ 2Q(1:30)1′ ILacZ 2 I(0.5)3 I(2.0)1 Blank′Key-Blank = SubstrateStd X = Standard of X ng/ml hGHXQLacZ Y = Sample Y obtained X days after transfection of a LacZ controlplasmid into 0.5 × 10(6) cells using the Qlagen kitXQ(1:Y)Z = Sample Z obtained X days after transfection of a 1:Y ratio ofDNA:Effectene reagent into 0.5 × 10(6) cells using the Qlagen kitXNegY′ = Sample Y obtained X days after transfection of no DNA into 0.8× 10(6) cells using the Qlagen kitXQ(1:Y)Z′ = Sample Z obtained X days after transfection of a 1:Y ratioof DNA:Effectene reagent into 0.8 × 10(6) cells using the Qlagen kitILacZ X = Sample X obtained one day after transfection of a LacZ controlplasmid into 0.7 × 10(6) cells using the Invitrogen kitI(X)Y = Sample Y obtained one day after transfection of Xug DNA into 0.7× 10(6) cells using the Invitrogen kitBlank′ = Buffer

Example 13 Immunophehenotypic Characterization of the EA1C-35 and Fa2N-4Cell Lines

Both the Ea1C-35 (passage 26) and Fa2N-4 (passage 30) cell lines werephenotyped by indirect immunofluorescence analysis using a panel ofantibodies against different hepatocyte or bile duct markers as well asagainst the SV40 immortalizing gene. The results from this analysis aresummarized in the Table 19 below: TABLE 19 Ea1C-35 Fa2N-4 Marker (%positive Cells) (% positive Cells) Albumin 90 100 Alpha Fetoprotein 0 0Connexin 32 50 80 CD 81 100 100 CD49f (integrin 0 0 alpha 6 chain) SV40T-antigen 100 100

The expression of connexin 32 was density dependent. At low platingdensity expression of these two proteins was undetectable. However whencells grew to confluent monolayers, a subpopulation of Ea1C-35 andfa2N-4 cells express this gap junctional protein which is only expressedby hepatocytes in adult liver tissue.

All cells expressed SV40 T-antigen, the immortalizing gene in theirnucleus. The well-differentiated nature of the immortalized liver cellsis indicated by the strong expression of the adult hepatocyte specificlineage markers, albumin and connexin 32 and the lack of the fetalhepatocyte marker, alpha fetoprotein. The cells do not express CD49f, abile duct marker. The cells express CD81, the putative receptor forhepatitis C virus glycoprotein-mediated viral infection. Aphotomicrograph of Fa2N-4 cells immunstained for CD81 is shown in FIG.35. Note that expression of CD81 is localized to the plasma membrane.

Taken together, all the above examples strongly indicate that the twoimmortalized human hepatocyte cell lines maintain many functionalattributes characteristic of hepatocytes in vivo and are an invaluablein vitro system to produce plasma proteins, including therapeutic plasmaproteins.

The inventions illustratively described herein can suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising,” “including,” “containing,” etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the future shown and described or anyportion thereof, and it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions herein disclosed can be resorted bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of the inventions disclosed herein.The inventions have been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thescope of the generic disclosure also form part of these inventions. Thisincludes the generic description of each invention with a proviso ornegative limitation removing any subject matter from the genus,regardless of whether or not the excised materials specifically residedtherein.

In addition, where features or aspects of an invention are described interms of the Markush group, those schooled in the art will recognizethat the invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. It is also to beunderstood that the above description is intended to be illustrative andnot restrictive. Many embodiments will be apparent to those of in theart upon reviewing the above description. The scope of the inventionshould therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent publications, are incorporated herein by reference.

1. A method of using immortalized human hepatocyte cells to produce aprotein comprising the steps of: (a) providing an immortalized humanhepatocyte cell that includes DNA that encodes and can express aprotein; (b) culturing the immortalized hepatocyte cell under conditionsin which a gene or genes encoding the protein are expressed so that theprotein is produced and processed in the immortalized hepatocyte cell;and (c) isolating the processed protein from the immortalized hepatocytecell; wherein the protein is expressed such that the protein isprocessed and glycosylated, if necessary, so that its in vivo functionis substantially preserved after its isolation.
 2. The method of claim 1wherein the protein is a plasma protein that is naturally produced byhuman hepatocytes.
 3. The method of claim 1 wherein the protein is aprotein that is not naturally produced by human hepatocytes.
 4. Themethod of claim 3 wherein the protein is a mutein of a protein that isnormally produced by human hepatocytes.
 5. The method of claim 1 whereinthe protein is a therapeutic protein.
 6. The method of claim 5 whereinthe therapeutic protein is a therapeutic plasma protein.
 7. The methodof claim 6 wherein the protein is selected from the group consisting ofFactor VIII, Factor IX, human growth hormone (hGH), α-1-antitrypsin, agrowth factor, muteins of Factor VIII, muteins of Factor IX, muteins ofhuman growth hormone, muteins of α-1-antitrypsin, and muteins of agrowth factor.
 8. (canceled)
 9. The method of claim 1 wherein theprotein is an IαIp protein.
 10. The method of claim 1 wherein theprotein is a protein selected from the group consisting of albumin,transcobalamin II, C-reactive protein, fibronectin, ceruloplasmin, otherproteins having structural, enzymatic, or transport activities, and amutein of a protein selected from the group consisting of albumin,transcobalamin II, C-reactive protein, fibronectin, ceruloplasmin, andother proteins having structural, enzymatic, or transport activities.11. (canceled)
 12. The method of claim 1 wherein the protein isexpressed by a gene that occurs naturally in the hepatocytes, andexpression of the naturally-occurring gene encoding the protein isenhanced by introduction of a high-level promoter into the hepatocytes.13. The method of claim 1 wherein expression is enhanced by introducingmultiple copies of the gene encoding the protein to be expressed, asubunit of the protein to be expressed, or a precursor of the protein tobe expressed via the use of one or more recombinant vectors thatinclude: (1) the gene encoding the protein to be expressed, a subunit ofthe protein to be expressed, or a precursor of the protein to beexpressed; and (2) at least one control element affecting thetranscription of the gene, the control element being operably linked tothe gene.
 14. The method of claim 13 wherein the recombinant vector isselected from the group consisting of SV40-derived vectors, murinepolyoma-derived vectors, BK virus-derived vectors, Epstein-Barrvirus-derived vectors, adenovirus-derived vectors, adeno-associatedvirus-derived vectors, baculovirus-derived vectors, herpesvirus-derivedvectors, lentiviral-derived vectors, retrovirus-derived vectors,alphavirus-derived vectors, and vaccinia virus-derived vectors.
 15. Themethod of claim 14 wherein the vector incorporates one or more reportergenes.
 16. The method of claim 1 wherein the expressed protein issecreted from the cell into the surrounding culture medium.
 17. Themethod of claim 1 wherein the protein is glycosylated.
 18. The method ofclaim 1 wherein the protein is processed post-translationally.
 19. Themethod of claim 1 wherein the protein is expressed in a form wherein itis fused to a cleavable tag.
 20. The method of claim 19 wherein thecleavable tag is selected from the group consisting of glutathioneS-transferase, the MalE maltose-binding protein, and a polyhistidinesequence.
 21. The method of claim 1 wherein the protein comprises atleast two different subunits, and wherein the immortalized hepatocytecell is transformed or transfected with at least two vectors, eachvector including: (1) DNA including at least one gene that encodes atleast one subunit of the protein; and (2) at least one control elementoperably linked to the DNA encoding at least one gene that encodes thesubunit of the protein.
 22. The method of claim 1 wherein theimmortalized human hepatocyte cell is virally immortalized.
 23. Themethod of claim 22 wherein the hepatocyte is immortalized bytransformation or transfection with substantially pure simian virus(SV40) DNA.
 24. The method of claim 23 wherein the substantially pureSV40 DNA encodes large T and small t antigens (Tag).
 25. The method ofclaim 1 wherein the immortalized human hepatocyte cell is derived fromprimary cryopreserved human hepatocytes.
 26. The method of claim 1wherein the hepatocyte includes tumor-suppressor-encoding DNA such thatsubstantially pure DNA encoding tumor suppressor can be isolated andpurified from the hepatocyte.
 27. The method of claim 1 wherein thehepatocyte includes DNA encoding Rb or p53 such that substantially pureDNA encoding Rb or p53 can be isolated and purified from the hepatocyte.28. (canceled)
 29. The method of claim 1 wherein the hepatocyte isnontumorigenic, has the ability to be maintained in a serum-free medium,and produces plasma proteins.
 30. The method of claim 1 wherein thehepatocyte is a hepatocyte of the Fa2N-4 cell line.
 31. The method ofclaim 1 wherein the hepatocyte is a hepatocyte of the Ea1C-35 cell line.32. A method of using eukaryotic cells, other than human hepatocytes, toproduce an IαIp protein comprising the steps of: (a) providing aeukaryotic cell, other than a human hepatocyte, that includes DNA thatencodes and can express proteins forming an IαIp protein complex, theeukaryotic cell having been transformed or transfected with at least onevector that includes: (1) DNA including at least one gene for aprecursor of a protein that is part of an IαIp protein; and (2) at leastone control element operably linked to the DNA encoding at least oneprecursor gene in order to enhance expression of the precursor gene; (b)culturing the transformed or transfected eukaryotic cell underconditions in which genes encoding proteins forming an IαIp protein areexpressed so that an IαIp protein is produced; and (c) isolating theexpressed IαIp protein from the transformed or transfected eukaryoticcell.
 33. The method of claim 32 wherein the eukaryotic cell is selectedfrom the group consisting of CHO cells, COS cells, and yeast cells. 34.The method of claim 32 wherein the eukaryotic cell is transformed ortransfected with two vectors: (1) a first vector that includes the genesH3 and AMBP; and (2) a second vector that includes the genes H2 and H1.35. An immortalized human hepatocyte cell that includes DNA that encodesand can express a protein, the immortalized human hepatocyte cell havingbeen transformed or transfected with at least one vector that includes:(1) DNA including at least one gene encoding a protein; and (2) at leastone control element operably linked to the DNA encoding the protein inorder to enhance expression of the protein, the protein being expressedby the cell such that the protein is processed and glycosylated, ifnecessary, so that its in vivo function is substantially preserved afterisolation of the protein.
 36. The cell of claim 35 wherein the proteinis a protein that is naturally produced by human hepatocytes.
 37. Thecell of claim 35 wherein the protein is a protein that is not naturallyproduced by human hepatocytes.
 38. The cell of claim 37 wherein theprotein is a mutein of a protein that is normally produced by humanhepatocytes.
 39. The cell of claim 35 wherein the protein is atherapeutic protein.
 40. The cell of claim 39 wherein the therapeuticprotein is a therapeutic plasma protein.
 41. The cell of claim 40wherein the protein is a therapeutic plasma protein selected from thegroup consisting of Factor VIII, Factor IX, human growth hormone (hGH),α-1-antitrypsin, a growth factor, muteins of Factor VIII, muteins ofFactor IX, muteins of human growth hormone, muteins of α-1-antitrypsin,and muteins of a growth factor.
 42. (canceled)
 43. The cell of claim 35wherein the plasma protein is an IαIp protein.
 44. The cell of claim 35wherein the protein is a protein selected from the group consisting ofalbumin, transcobalamin II, C-reactive protein, fibronectin,ceruloplasmin, other proteins having structural, enzymatic, or transportactivities, and a mutein of a protein selected from the group consistingof albumin, transcobalamin II, C-reactive protein, fibronectin,ceruloplasmin, and other proteins having structural, enzymatic, ortransport activities.
 45. (canceled)
 46. A method of treating a diseaseor condition comprising the steps of: (a) providing an active proteinproduced according to the method of claim 1; and (b) administering theactive protein to a patient suffering from the disease or condition in atherapeutically active quantity to treat the disease or condition. 47.The method of claim 46 wherein the disease or condition is a disease orcondition affecting the liver.
 48. The method of claim 47 wherein thedisease or condition affecting the liver is selected from the groupconsisting of sepsis, cancer, hepatitis, and liver failure.
 49. Themethod of claim 46 wherein the disease or condition is a disease orcondition affecting an organ other than the liver.
 50. The method ofclaim 49 wherein the disease or condition is selected from the groupconsisting of cancer, joint inflammation, and arthritis.
 51. Apharmaceutical composition for treating a disease or conditioncomprising: (a) an IαIp protein produced by eukaryotic cells in aquantity therapeutically effective to treat a disease or condition; and(b) a pharmaceutically acceptable carrier.
 52. The pharmaceuticalcomposition of claim 51 wherein the disease or condition is a disease orcondition affecting the liver.
 53. The pharmaceutical composition ofclaim 52 wherein the disease or condition affecting the liver isselected from the group consisting of sepsis, cancer, hepatitis, andliver failure.
 54. The pharmaceutical composition of claim 51 whereinthe disease or condition is a disease or condition affecting an organother than the liver.
 55. The pharmaceutical composition of claim 54wherein the disease or condition is selected from the group consistingof cancer, joint inflammation, and arthritis.
 56. The method of treatinga disease or condition of claim 46 wherein the active protein is anactive plasma protein.
 57. (canceled)
 58. (canceled)
 59. (canceled) 60.(canceled)
 61. The method of claim 56 wherein the active plasma proteinis selected from the group consisting of Factor VIII, Factor IX, humangrowth hormone (hGH), α-1-antitrypsin, and a growth factor.